Patent Application: US-3657498-A

Abstract:
the present invention relates generally to the use of genetic engineering to induce developmental regulation in anther tissue of plants , and more particularly to induce nuclear male sterility , and to genetic sequences useful for same . more particularly , the present invention relates to the identification of a genomic clone and promoter capable of tissue - and development - specific expression which provides a means of tissue and developmental regulation in plants and more specifically a means of producing nuclear male sterile plants . even more particularly , the present invention provides a genomic clone having a nucleotide sequence as set forth in seq id no . 1 or homologous sequences thereof such as the nucleotide sequence as set forth in seq id no . 3 .

Description:
constriction of genonic library , screening and isolation of the genomic clone bgp1 . a genomic library was prepared from leaf material of brassica campestris cv . t15 . genomic dna was isolated according to standard procedures ( murray et al ., 1980 ) and partially digested with sau 3a . sau 3a fragments were size fractionated on a glycerol gradient ( 10 - 40 %) by centrifugation at 40 , 000 rpm overnight . aliquots of 500 μl fractions were taken and diluted 1 : 2 in te buffer ( 10 mm tris - hci , ph8 . 0 ; 1 mm edta ). dna from the chosen fractions was then recovered by precipitation with ethanol and centrifugation at 13 , 000 g for 30 minutes . the resultant 9 - 23 kb fragments were ligated into embl3 bam h1 arms ( stratagene ). the ligation mix was then packaged into phage using packagene ( promega biotec .) to yield the genomic library . the library was plated on lb media at a density of approximately 10 , 000 plaques per 90 mm plate using escherchia coli nw2 ( woodcock et al ., 1988 ) as the host strain . duplicate plaque lifts were performed using hybond - c extra following the manufacturer &# 39 ; s protocol . the filters were hybridized with 32 p - labelled bgp1 in 2 × sspe , 0 . 5 % w / v blotto , 1 % w / v peg 20 , 000 , 7 % w / v sds and 250 mg / ml ( final volume ) denatured herring sperm dna at 65 ° c . filters were washed at 65 ° c . for 30 minutes in 2 × ssc , 0 . 1 % w / v sds and for 15 minutes in 0 . 2 × ssc , 0 . 1 % w / v sds . filters were exposed to kodak x - omat film overnight at − 70 ° c . the genomic clone obtained is designated “ bgp1 ”. a series of overlapping deletion clones ( bgp1 . 1 - bgp1 . 7 ) were generated from the bgp1 4 . 2 kb hindiii genomic fragment by digestion with exonuclease iii and religation . the protocols supplied with the nested deletions kit ( pharmacia lkb ) were followed . southern blot analysis demonstrated that bgp1 . 1 - bgp1 . 5 but not bgp1 . 6 and bgp1 . 7 show homology to bgp1 . sequencing then commenced using bgp1 . 1 though bgp1 . 5 as templates . sequencing reaction was performed on double - stranded template according to the t7 polymerase sequencing kit manual ( pharmacia lkb ). both strands were sequenced using t7 , sp6 or synthetic primers made to internal sequences . rna gel blot hybridizations were performed using total rna ( 20 μg per lane ) separated by electrophoresis on formaldehyde - agarose gels and blotted onto hybond - n ( amersham ) nylon filters ( maniatis et al ., 1982 ). filters were prehybridized , hybridized with 32 p - labelled oligonucleotide and washed according to the manufacturers specifications ( amersham ). dna gel blots were performed using 10 μg of b . campestris dna per digest separated on 0 . 8 % w / v agarose gels and blotted onto hybond - n filters following standard protocols ( maniatis et al ., 1982 ). prehybridization , hybridization with 32 p - labelled dna and washing was again done according to the manufacturer &# 39 ; s specifications ( amersham ). the transcriptional start point of bgp1 was determined by primer extension analysis performed according to standing procedures ( maniatis et al ., 1982 ). a 15 - mer synthetic oligonucleotide of sequence 5 ′- cgttttggcgaccca - 3 ′ ( seq id no . 11 ) complementary to nucleotides 22 - 36 of bgp1 was end - labelled with [ g 32 p ] atp ( amersham ) and t4 polynucleotide kinase ( promega biotech ). after annealing and extension of the primer , the products were analysed on a polyacrylamide sequencing gel . the 0 . 8 kb pst i - hae iii dna fragment ( from position − 767 to + 100 including the 0 . 7 kb 5 ′ flanking region and 100 nucleotides of the 5 ′ untranslated leader sequence of the bgp1 gene ), was excised from bgp1 . 3 , a deletion clone of bgp1 , and ligated to bluescript (+) ks ( stratagene ). this fragment was then excised as a hindiii - bam hi fragment and inserted into the polylinker of the vector , pbi 101 ( stratagene ). this vector is a derivative of the binary vector pbin 19 ( bevan , 1984 ) which contains a promoter - less gus gene cassette ( jefferson et al ., 1986 ) fused to the nopaline synthase polyadenylation region . the resulting bgp1 - gus chimeric construct , designated as pbgp1 . 2 , was mobilized into agrobacterium tumefaciens strain lba 4404 , by conjugating with a helper plasmid prk 2013 ( koncz and schell , 1986 ). ( i ) tobacco : leaf discs of nicotiana tabacum var . wisconsin 39 were transformed with agrobacterium tumfaciens essentially as described in horsch et al . ( 1985 ). shoots arising from leaf discs were rooted on ms medium ( gibco laboratory ) containing 1 . 0 μg / ml iaa , 1 . 0 μg / ml bap , 100 μg / mlkanamycin , transferred to soil and grown to flowering in the greenhouse . arabidopsis thaliana var landsberg roots were transformed according to valvekens et al . ( 1985 ). transgenic plants were selected on medium containing 50 μg ml − 1 kanamycin . histochemical gus assays were performed essentially as described by jefferson et al . ( 1987 ). for histochemical assay , plant materials were placed in the wells of a microtiter plate containing 1 mm x - glu ( 5 - bromo - 4 - chloro - 3 - indolyl - β - d - glucuronic acid , sigma ) in 0 . 1 m napo 4 ph 7 . 0 , 0 . 1 % v / v triton - x 100 and incubated at 37 ° c . for 8 to 12 h . in arabidopsis , the positively stained flowers were processed for cryo - sectioning . the samples were embedded , rapidly frozen in tissue - tek oct compound ( miles inc ., elkhart , ind .) and sectioned at − 20 ° c . using a 2800 frigocut cryostat ( reicher - jung , germany ). the developmental stages of anthers were determined by staining the sections with dapi ( coleman and goff , 1984 ). a genomic clone , designated bgp1 was isolated from brassica campestris using cdna clone bcp1 as a probe . a partial restriction endonuclease site analysis of the 11 kb genomic fragment in bgp1 resulted in the map presented in fig1 . southern blot analysis using bcp1 as a probe revealed that a 4 . 2 kb hind iii fragment from the 11 kb genomic clone contained the coding region of the gene . this fragment was then subcloned and partially sequenced . a total of 1620 bp of dna was determined , this included the entire sequence of the coding region along with 767 bp of 5 ′ flanking sequence . comparison of the cdna sequence of bcp1 ( theerakulpisut et al ., 1991 ) with the coding region of genomic clone bgp1 revealed an overall homology of 88 %. no introns are present . the sequence of the bgp1 coding region , 767 bp of 5 ′ flanking region and 392 bp of 3 ′ flanking region is presented in fig1 . in order to determine the expression pattern of bgp1 gene , a bgp1 - specific oligonucleotide was synthesized based on the sequence between nucleotides 181 - 201 ( 5 ′- ggctgctaccgtaaccgatgt - 3 ′ [ seq id no . 10 ]) ( fig1 ), a region which shows a high level of variability between the two genes , bgp1 and bcp1 . this 21mer oligonucleotide was used to probe a northern blot containing total rna isolated from b . campestris pollen , leaf , stem , and flower ( minus anther ). as a negative control bcp1 dna was included on the blot to ensure the specificity of the oligonucleotide . fig2 shows that the bgp1 - specific oligonucleotide hybridizes to rna present in pollen but not to rna present from any other tissue tested . the size of the transcript = 700 nucleotides is approximately the same size as the rna transcript to which clone bcp1 hybridizes ( theerakulpisut et al ., 1991 ). to determine whether the clone bcp1 represents a transcript from a member of a gene family , the cdna insert was used to probe a dna gel blot of total b . campestris dna . fig3 shows that bcp1 cdna insert hybridizes to several genomic bands including the 4 . 2 kb hind iii fragment representing the bgp1 gene . it is difficult to estimate the gene family copy number from this blot but there are at least two members in the gene family . the transcriptional start point of bgp1 was determined by primer extension analysis . an oligonucleotide was synthesized based on the sequence between nucleotides 22 - 36 ( fig1 ). fig4 shows that when this primer was used in extension analysis and the labelled products run next to the sequence of clone bgp1 , a fragment of length 61 nucleotides can be detected . this indicates that the a nucleotide at position 1 ( fig1 ) is the first nucleotide transcribed from the bgp1 gene . fainter bands are likely to be due to homologous transcripts from other members of the bcp1 gene family . the promoter region of the bgp1 gene contains a tata box sequence ( caactatatatag [ seq id no . 12 ]) which is located 26 nucleotides upstream of the start of transcription ( see fig1 ). in agreement with the “ scanning mechanism ” theory ( kozak , 1986 ), the translational start of the bgp1 gene is the first atg codon present in the sequence , which is found 69 nucleotides downstream from the start of transcription ( fig1 ). the putative start codon gives the longest possible open reading frame ( 357 nucleotides ) and its flanking regions match perfectly with the consensus proposed for plant genes of aacaatggc [ seq id no . 13 ] ( lutcke et al ., 1987 ). the resulting untranslated leader sequence is characteristically a / t rich ( 63 %). the predicted protein sequence of clone bgp1 is shown under the nucleotide sequence in fig1 . the bgp1 protein sequence is very similar to the predicted protein sequence from bcp1 ( theerakulpisut et al ., 1991 ) showing 87 . 5 % identity . a computer search comparing the nucleotide and amino acid sequence of clone bgp1 with the sequences contained in genbank , embl and nbrf databases revealed no significant homologies . the 5 ′ upstream region of clone bgp1 was examined for homology to the promoter regions of both anther and pollen specific genomic clones . a comparison of the entire 767 bp bgp1 5 ′ region was made with a range of 5 ′ sequences from pollen / anther specific genes ( hamilton et al ., 1989 ; twell et al ., 1989 , 1991 ; koltunow et al ., 1990 ; albani et al ., 1991a , 1991b ). no significant regions of homology were observed . transformation of the full length promoter construct into arabidopsis — high levels of gus activity present in pollen and tapetum the histochemical distribution of the gus activity driven by the 767 bp bgp1 5 ′ region carried by the construct pcb12 is illustrated in fig5 . fig5 a shows that high levels of gus activity were present in anthers , but not in petals , sepals , filaments and pistils . no gus activity was detected in anthers of control untransformed plants . the developmental pattern of bgp1 - promoter activity was also analysed in transgenic plants carrying the construct pcb12 . in cryosections of developing arabidopsis anthers containing an intact tapetum , fig5 b and 5c show that high levels of gus activity were present in the tapetum , whereas only low levels were detactable histochemically in the pollen at early bicellular stage . in near mature anthers , in which the tapetum had begun to degenerate , fig5 d shows that high levels of gus activity were present both in the degenerating tapetum and pollen grains . fig5 f and 5g show that very high levels of gus activity were present in mature pollen , but not in other tissues of the anther . fig5 e and 5h show that tissues of control untransformed anthers and pollen produced no histochemically detectable levels of gus activity . in transgenic tobacco plants carrying pcb . 2 , gus activity was detected in pollen ( fig5 i ). in addition gus activity was tested in anther sections taken from flower buds at several developmental stages . in tobacco , flower bud length correlates well with gametophytic development ( koltunow et al ., 1990 ). sections were taken from flower buds of sizes 3 mm ( tapetum formation commences ), 4 mm ( tapetum and pollen sacs distinct ), 5 mm ( meiosis begins ), 6 mm ( tapetum large and multinucleate ), 7 mm , 8 mm ( meiosis complete ) through to 14 mm ( tapetum shrunken , pollen grains begin to form ). no gus activity was detected in the tapetum at any of these developmental stages . to identify cis - acting elements controlling the temporal and spatial expression pattern of bgp1 a series of 5 ′ deletion clones were created . these constructs , shown in fig6 were transferred to arabidopsis thaliana by agrobacterium tumefaciens mediated transformation . gus activity was analysed on primary transformants . at least 10 individual transformants were analysed for each construct . the gus expression pattern for each of the constructs is presented alongside each of fig6 . deletion of the full length promoter down to − 580 ( pcb13 ) abolished any detectable gus expression in the pollen of 87 % of the plants tested . expression in the tapetum of plants carrying pcb1 . 3 was unaffected . however , if further deletion removed the region between − 322 and − 580 ( pcb1 . 4 ), gus expression in the pollen was restored in all the plants tested . progressive 5 ′ deletions down to − 260 ( pcb1 . 5 ) and − 168 ( pcb1 . 6 ) gave the same result , gus expression was observed in both the tapetum and the pollen . the smallest construct tested however , which contained only the 5 ′ region up to position − 116 , directed gus expression in the tapetum only . a bcp1 antisense gene was constructed by inserting the cdna clone bcp1 in the reverse orientation between an anther - specific promoter , bgp1 and nopaline synthase ( nos ) sequence . it was then cloned into the plant transformation vector , bin 19 ( fig8 ). the resulting construct was mobilised to agrobacterium tumefacien strain lba 4404 and introduced into arabidopsis thaliana var landsberg using standard procedures ( valvekens et al ., 1988 ). the transgenic plants carrying the antisense construct were selected by kanamycin resistance . the transgenic plants were examined for male - fertility in terms of the number of seeds produced following self - pollination . a total of 50 flowers from each of 20 different healthy plants were examined . all the plants produce phenotypically normal flowers . however , the plant produced short siliques typical of male sterile plants ( moffat and sommerville , 1988 ) and no seeds were set after selfing ( fig9 ). the transgenic plants carrying the bcp1 antisense construct were examined for their female - fertility by cross - pollinating with pollen from wild - type plants . ten flowers from three transgenic plants were pollinated with wild - type pollen . all the flowers produced normal elongated siliques following cross - pollination , indicating that female function is normal in these antisense transgenic plants . pollen grains from both wild - type and antisense plants were examined by scanning electron microscopy for possible alteration of pollen surface structure and morphology . pollen from five of the antisense plants appeared to be collapsed and shrunken when prepared a similar way to wild - type pollen . some of the pollen grains showed aberrant exine structure . in one antisense plant , the formation of the fish - net patterned ektexine was irregular , with prominent patches where the ektexine was missing over the pollen surface . in the antisense plants , light and transmission electron microscopic analyses showed that in mature pollen , the internal protoplasmic structure was completely disorganised or empty . developmental studies showed that tapetal and microspore differentiation was normal until the time of first pollen mitosis . the cytoplasm of the pollen grains then developed multiple vacuoles , and became disorganised . these data indicate that sterility of the pollen grains sets in at about the time of maximal expression of the gene bcp1 in the pollen grains . while tapetal development appeared normal , the 100 % effectiveness of the antisense construct in all 20 plants suggests that expression of bgp1 in the tapetum is vital for normal pollen development . pollen grains from wild - type and antisense plants were examined for viability using the fluorochromatic reaction ( fcr ) test ( heslop - harrison et al ., 1984 ). pollen from wild - type gave 99 % positive reaction , indicating high pollen quality , whereas pollen from antisense plants showed no positive fcr staining , indicating that pollen quality has been lost , and membrane integrity has been detrimentally altered . these data show that bgp1 gene is essential for normal pollen development . this is shown by the male sterility induced when the gene is present in antisense rna version . bgp1 is expressed in both the tapetum and pollen , and down regulation of its expression in the antisense plants clearly shows the importance of the gene product for normal development . rna gel blot studies indicated that a gene homologous to b . campestris bgp1 is expressed in arabidopsis thaliana ( fig1 ). the specificity and pattern of expression in anthers of arabidopsis bgp1 was isolated by screening an arabidopsis genomic library with the brassica bgp1 cdna clone ( fig1 ). dna sequencing studies show that the arabidopsis bgp1 cdna genomic clone in 1132 bp , with an orf of 137 amino acids ( compared with 119 in brassica ). the deduced amino acid sequence does not contain introns and encodes an alanine - rich ( 16 %) protein with a relative molecular mass , m r 14k ( compared with 12k brassica ). the nucleotide and deduced amino acid sequences of bgp1 show no homology with other known genes or proteins in the databases . no potential n - glycosylation sites are present in the amino acid sequence . mouse polyclonal antibodies raised against two synthetic peptides based on hydrophylic regions of the brassica bgp1 amino acid sequence recognised m r 11 - 12 k polypeptides by western analysis . accordingly , the results indicated that the bgp1 gene is expressed specifically in tapetum and pollen and encodes a protein of m r 12 - 14k in both brassica species and arabidopsis species . to demonstrate that 5 ′ sequences control bgp1 gene developmental specificity , the escherichia coli gus gene was fused with a 0 . 77 kb upstream fragment ( nucleotides − 767 to + 100 ; xu et al ., 1993 ), containing the start codon and then transformed arabidopsis plants with the chimaeric bgp1 gus gene . several independent transformants were obtained . each transformant showed gus enzyme activity in both tapetum and pollen . the pattern of gus activity in anthers of transgenic plants is consistent with the expression of endogenous bgp1 gene in brassica and arabidopsis . comparison of 5 ′- flanking regions of bgp1 from both brassica and arabidopsis shows that the two genes share a conserved region of high homology in the 167 nucleotides that lie immediately upstream of the transcriptional initiation site ( fig1 b ). there is no significant homology between the 5 ′ regions of the two genes beyond this point . because of the highly conserved pattern of expression of this gene in anthers of the two genera , it was expected that this 167 bp 5 ′ region may be sufficient to direct the normal developmental expression of the genes . to examine this , a chimaeric gene was constructed by fusing the 167 bp fragment with gus ( nucleotides − 167 to + 100 ; xu et al ., 1993 ). the arabidopsis plants transformed with this construct showed the same pattern of gus enzyme activity in anthers as those transformed with larger promoter fragments . since gus enzyme activity in plants transformed with a truncated 5 ′ fragment appeared to be relatively less than those with the larger fragment , it was decided that the region upstream of the − 167 bp may have an enhancer effect for bgp1 gene expression . the 0 . 77 kb bgp1 gene regulatory fragment was fused with antisense bcp1 cdna expression . this chimaeric construct was introduced into arabidopsis plants and 22 primary transformants ( t 0 ) were obtained . the transformants appeared identical to untransformed control plants with respect to growth rate , height , leaf and flower morphology , time of flowering and flower colour ( fig1 ). however , 7 of antisense transformants failed to show elongation of siliques , indicating loss of fertility . microscopic examination of flowers of antisense transformants ( t 0 ) showed the presence of defective pollen grains in the anthers , confirming that the effect is specifically on male rather than female fertility . pollen from anthers of antisense transformants was negative when tested for pollen quality by fcr test ( heslop - harrison et al ., 1984 ) compared with pollen from anthers of normal plants ( fig1 ). use of alexander &# 39 ; s stain ( which indicates the presence or absence of cytoplasm in pollen grains as a measure of sterility ) showed that & gt ; 90 % of pollen in antisense transformants is present as empty exines ( green staining ), while the remaining grains had cytoplasm ( weak pink or red staining ) in various stages of degeneration ( fig1 ). in contrast , pollen from anthers of normal plants showed densely staining ( purple ) grains . sections of anthers were prepared from both transformed and untransformed ( normal ) plants . male sterile anthers showed collapsed pollen sacs , and pollen grains without visible cytoplasmic contents ( fig1 a ). rare grains showed some residual cytoplasm that appeared disorganised and lysed ( fig1 a ). all other anther tissues and cell types appeared identical to normal anthers . dna gel blot analysis of the male sterile primary transformants showed that the male sterility phenotype is linked with the presence of the antisense cdna in their genome ( fig1 a ). the presence of the antisense insert was tested both by use of bgp1 cdna and neomycin phosphotransferase ( nptii ) as hybridisation probes . to determine whether the male sterility is a stably inherited trait , antisense transformants were crossed with pollen from normal ( untransformed ) plants . normal silique formation and seed set occurred in all cases . these results indicate that antisense transformants are male sterile , their pistils are able to recognise and transmit pollen normally , and female fertility is unaffected . eight of t 1 plants were analysed and all inherited the male sterility phenotype . in 4 of 8 t 1 plants , the presence of antisense bgp1 gene was further analysed by dna gel blot analysis ( fig1 b ). the male sterile phenotype and presence of antisense insert completely co - segregated . the introduced gene is present in the genomic dna of the analysed t 1 plants with male sterile pollen and absent in the genome of t 1 plants with normal viable pollen . inheritance of male sterility phenotype is also observed in t 2 generation . the expression of both endogenous and antisense mrna expressing bgp1 in both primary transformants was studied and male sterile t 1 plants ( fig1 c ). with sense - specific probe , a single mrna band of approximately 700 bp was detected in flowers of untransformed plants . the presence of sense bgp1 mrna could not be detected in either primary transformed or t 1 plants with male sterility phenotype . however , using an antisense - specific probe , a strongly hybridising transcript of approximately 750 bp was detected in flowers of all male sterile transformants . no antisense rna was detected in control untransformed plants . thus , the male sterility phenotype is linked with high expression of antisense bcp1 mrna and loss of sense bcp1 mrna . in order to define the stage of pollen development when arrest is initiated , thin sections of developing anthers of both normal plants and antisense transformants were prepared . at tetrad and uninucleate microspore stage , both tapetal cells and microspores appeared normal in both types of anthers . this is the stage when tapetum is most active and the exine is completely formed . at the late microspore stage , the microspore cytoplasm showed signs of vacuolation and autolysis ( fig1 ) which appeared to be complete before microspore mitosis . this rapid loss of cellular contents ultimately results in complete collapse of the microspores , which appear as empty shells ( fig1 ), since the exine remains unaffected . the tapetum appeared normal in both types of anther . this developmental sequence was similar in both primary transformants ( t 0 ) and male sterile t 1 generation . 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 of any two or more of said steps or features . albani d , robert l s , donaldson p e , altosaar i , arnison p g , fabijanski s f ( 1991a ) plant mol . biol . 15 : 605 - 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verlag , n . y ., pp 97 - 125 . moffatt b a and sommervile c ( 1988 ) plant physiol . 86 : 1150 - 1154 . murray h g , thompson w f ( 1980 ) nucl . acids res . 8 : 4321 - 4325 . sambrook et al . ( 1989 ) molecular cloning : a laboratory manual . cold spring harbour laboratory press , new york . theerakulpisut p , xu h l , singh m b , pettitt j m , knox r b ( 1991 ) plant cell . 3 : 1037 - 1084 . twell d , wing r a , yamaguchi j , mccormick s ( 1989 ) mol . gen . genet . 217 : 240 - 245 . twell d , yamaguchi j , wing r a , ushiba j , mccormick s ( 1991 ) genes dev . 5 : 496 - 507 . valvekens d , van montagu m , van lijsebettens m ( 1988 ) proc . natl . acad . sci . 85 : 5536 - 5540 . woodcock d m , crowther m , diver w d , graham m , bateman c , baker d j , smith s s ( 1988 ) nucl . acids res . 16 ; 4465 - 4482 . xu h , davies s p , kwan b v h , o &# 39 ; brien a p , singh , m b , knox r b ( 1993 ) mol . gen . genet . 239 : 58 - 65 . atg ggt cgc caa aac gct gtc gta gtt ttt ggc ctt gtg ttc ttg gcc 48 atc ctt ggc ctc gcc gca gct gcc tcc tct ccg tct cct tca gcg tca 96 ccc tcc aaa gct ccg gct gct acc gta acc gat gtc gaa gct cca gtg 144 agc gag gac acc att gga acc acc gat gac gat gca gct gct tct cca 192 ggt gat ggt gac gta gct gtg gct ggt cct cta gga agt gac tcc tcc 240 tac ggt agt aat gga cct tca cct tct act gat gct gct gac agc ggc 288 gcg cct gct ctt ggc gtc tct gcg gtc ttc gtt ggt gtt gca tcc atc 336 atg ggt cgc caa aac att gtc gtc gtg gtt gcc ctc gtc ttc atc cgg 48 atc att ggc ctt gcc gca gct gcc tcc tct cca tct cct tca gcg tct 96 ccc tcc aaa gct cca gct gcc tcc aaa acc gat cat gtc gag gct cca 144 gtc acc gat gac caa atc gga acc acc gat gac gat gca gct cct act 192 cct ggt gac ggt gac gtt gca gtg gct ggt cct cta gga agt gac tcc 240 tcg tac gac aat gcc gct aca ggc tct gct gat tct gcc aaa agc ggt 288 gcg gca gct ctt ggc gtc tct gcg gtc gtc gtt ggt gtt aca tca ttg 336 ctg gtt ctt tct tgt tac tca agt tgg gca ttg ttt tat gat aag aag 384