isolation of the anther - specific genes a9 and a3 from arabidopsis thaliana anther - specific cdnas were isolated by differential screening of brassica napus cdna libraries constructed from rna extracted from dissected anthers , as described below . cdna clones a3 and a9 were isolated from a library constructed from anthers that were 1 . 4 - 1 . 8 mm in length . this library was constructed in the vector lambda zap ( stratagene ). the a3 and a9 cdnas were used as probes to isolate homologous genes from an a . thaliana genomic library constructed in the vector lambda dash ( stratagene ). plant material . all seeding material for nucleic acid isolation was obtained from 2 - 3 week old plants grown in a controlled environment growth cabinet with 18 h photoperiod at 24 ° c . seedling rna for differential screening and northern blot analysis was obtained from b . napus oleifera var &# 34 ; topaz &# 34 ;. male fertile buds were collected from field grown plants of b . napus oleifera var . &# 34 ; lictor &# 34 ; ( nickersons seeds , cambridge , uk ). male - sterile buds were obtained from field grown b . napus var . cms &# 34 ; ogura &# 34 ; ( nickersons seeds , cambridge , uk ) plants . dissection of anthers . for cdna library construction , flower spikes were quickly harvested and kept at 4 ° c . until required , but no longer than 5 h . anthers were dissected from appropriately sized buds using fine forceps and immediately frozen in liquid nitrogen . pollen isolation . microspores were isolated from fresh buds of the appropriate length using the method of choung and beaversdorf ( plant sci . 39 219 - 226 ( 1985 )), and frozen at - 80 ° c . prior to rna isolation . ( pollen isolated from frozen buds yielded only highly degraded rna ). collection of buds . large samples of complete whorls of buds , at a stage immediately prior to the opening of first flowers , were frozen in liquid nitrogen and stored at - 80 ° c . cytological staging of anthers and buds . the developmental stage of buds of predetermined length was assessed by light microscopic examination of sporogenous cells , microspores or pollen grains extruded from whole anthers squashed in the presence of aceto - orcein or acridine orange . accurate determination of bud length was performed using a low - powered light microscope equipped with a calibrated eyepiece graticule . bud lengths stated were measured from the base of the pedicle to the tip of the outermost sepal . rna isolation and analysis . material intended for low resolution northern dot blot analysis or for mrna isolation was ground to a fine powder in a mortar cooled with liquid nitrogen . total rna was isolated from the powder using a phenol based method as described previously ( draper et al ., &# 34 ; plant genetic transformation and gene expression : a laboratory manual &# 34 ;, blackwell scientific publishers , oxford ( 1988 )). poly ( a ) + rna was purified by two rounds of oligo ( dt )- cellulose chromatography essentially as described in the maniatis et al manual . rna for high resolution dot blots was isolated according to the method of verwoerd et al ., nuc . acids res . 17 2362 ( 1989 )). cdna library construction and screening . cdnas were synthesised from poly ( a ) + rna using ( amenham or pharmacia ) cdna synthesis kits , according to the manufacturers instructions . cdnas were ligated into ecori cleaved dephosphorylated lambda zap i ( stratagene ) (&# 34 ; sporogenesis &# 34 ; library ) or lambda zap ii ( stratagene ) (&# 34 ; microspore - development &# 34 ; library ) and packaged using amersham in vitro packaging extracts . clones were screened differentially , on duplicate hybond - n filters ( amersham ) with 32 p !- labelled single - stranded cdna probe prepared from either the appropriate anther poly ( a ) + rna or seedling poly ( a ) + rna according to sargent methods in enzymol . 152 423 - 432 ( 1987 )). ( the expression hybond - n is a trade mark .) rna dot and gel blots . total rna for dot - blots was spotted onto hybond n ( amersham ) according to the manufacturers instructions . northern gels were run and rna transferred to hybond - n according to fourney ( brl focus 10 5 - 7 ( 1988 )). hybridisation and washing of hybond - n filters was according to manufacturers instructions . in situ hybridisation . for embedding and sectioning b . napus buds were frozen in cryo - m - bed ( taab laboratories equipment ltd ). ( the expression cryo - m - bed is a trade mark .) sections were cut nominally 10 μm thick , mounted on subbed slides ( van prooijen - knegt et al ., histochemical j . 14 333 - 344 ( 1983 )) fixed in 4 % paraformaldehyde and dehydrated . 35 s ! rutp (& gt ; 1000 ci / mmol , amenham sj . 1303 ) labelled sense and anti - sense rna probes were transcribed from the t3 and t7 promoters of bluescript sk - ( stratagene ), in which the cdnas are cloned . ( the expression bluescript sk - is a trade mark .) following transcription , probes were cleaved by alkaline hydrolysis to generate probe fragments approximately 150 bp in length . the hybridisation solution was 50 % formamide , 300 mm nacl , 10 mm na 2 hpo 4 ph 6 . 8 , 10 mm tris - hcl ph 7 . 5 , 5 mm edta , 0 . 02 % bovine serum albumin , 0 . 02 % ficoll , 0 . 02 % polyvinylpyrrolidone , 10 mm dithiothreitol , 10 % dextran sulphate , 0 . 7 mg / ml e . coli trna , 50 - 100 ng / ml probe stock ( 6 . 7 × 10 5 cpm / ng probe ). sections were hybridised in 30 μl hybridisation solution at 50 ° c . for 16 h . slides were washed 3 × 1 h at 50 ° c . in 50 % formade , 300 mm nacl , 10 mm na 2 hpo 4 ph 6 . 8 , 10 mm tris - hcl ph 7 . 5 and then rinsed in rnase a buffer to remove formamide . rnase a treatment , ( 150 μg / ml rnase a in 500 mm nacl , 10 mm tris hcl ph 7 . 5 ), was carried out at 37 ° c . for 1 h . the slides were then washed twice in 2 × ssc ( 0 . 3m nacl , 0 . 03m na citrate , ph 7 . 0 ) at 65 ° c . for 30 min , dehydrated through graded alcohols and dried . for autoradiography , slides were dipped at 45 ° c . in ilford k5 nuclear track emulsion ( 1 g / ml in 1 : 59 glycerol : water mix ). ( the expression ilford k5 is a wade mark .) exposure time was between 2 and 14 days . development was in kodak d19 . ( the expression kodak d19 is a trade mark .) following development sections were stained with methylene blue and made permanent . northern hybridisation analysis using rna extracted from b . napus anthers , pollen , carpels and seedlings indicated that a3 was only expressed in anthers of length 1 . 6 - 2 . 3 mm with maximal expression between 1 . 8 - 2 . 3 mm . thus a3 temporal expression spans the period in anther development when the microsporocytes are in meiotic division to early microspore interphase . in situ hybridisations suggest that in b . napus , a3 is expressed solely in the tapetum of the anther . the a3 cdna is 347 bp in length and contains an open - reading frame ( orf ) extending from position 1 - 329 bp ( fig1 ) suggesting that this clone is not full - length . the estimated size of b . napus a3 mrna from northern gel blots is about 500 bp , and again suggesting that this clone is not full length . the a3 cdna was used to isolate homologous cdna clones ( e3 and e5 ) from the same b . napus library . e5 cdna is 422 bp long and contains an orf from position 1 - 333 ( fig2 a ). this cdna is identical to a3 cdna over the region they overlap and extends the a3 sequence 5 bp 5 &# 39 ; of the start of a3 and 69 bp 3 &# 39 ; of the end of a3 . the e3 cdna is 398 bp in length with an orf extending from position 1 - 314 bp ( fig2 a ). the e3 cdna is 95 % identical to the e5 cdna at the nucleotide level and the putative orf products are 91 % identical . the cloning of e5 which is homologous but not identical to a3 is evidence that probes based on the sequence of the anther - specific gene a3 would enable the cloning of homologous anther - specific genes . a 15 kb a . thaliana genomic clone ( g3 . 6 ) was isolated that hybridises to a3 cdna ( fig3 ). the 2300 bp hindiii - hincii region which hybridised strongly with the a3 probe , was subcloned from g3 . 6 and partially sequenced ( fig4 ) revealing an orf ( positions 770 - 1126 ) that is highly homologous to the b . napus e5 cdna ( 82 % at the nucleotide level and 76 % at the protein level ) ( fig2 a , b ). the a . thaliana a3 gene does not apparently contain introns . the putative protein encoded by a3 consists of 118 amino - acids with a molecular mass of 12 . 9 kda . a search of the nbrf protein database ( release 34 ) did not reveal any proteins homologous to the putative a3 protein . there is a tata box consensus sequence ( joshi , 1987 ) between positions 699 - 707 bp , 63 bp upstream of the putative start of the a3 coding region . northern analysis and in situ hybridisations indicate that the b . napus a9 gene is expressed in the tapetal cells of anthers of length 1 . 5 - 2 . 3 mm , with maximal expression between 2 . 0 - 2 . 3 mm . expression of a9 is initiated in anthers that contain meiocytes and continues into anthers that contain microspores in early first interphase . the a9 cdna is 490 bp in length containing an orf from position 1 - 296 bp ( fig5 ). from northern gel blots , the estimated size of the a9 mrna in b . napus is about 550 - 600 bp . the abundance of the a9 mrna was estimated at between 0 . 1 - 0 . 2 % of total anther polya + mrna . a 13 kb a . thaliana genomic clone ( g9 . 1 ) was isolated that hybridised to the a9 cdna ( fig6 ) and a 3145 bp xbai fragment cloned and partially sequenced ( fig7 ). this fragment contains an orf at position 1461 - 1781 that is 76 % identical to the a9 cdna orf at the nucleotide level ( fig8 a ) and the putative products of these orfs are 73 % identical ( fig8 b ). comparison of the cdna and genomic sequences suggests that the orf in the cdna starts at position 9 bp ( fig5 ) and that the a9 gene contains no introns . 70 bp upstream of the putative start of the a9 gene ( positions 1382 - 1389 bp ) is a tata box conforming to the consensus sequence of joshi ( 1987 ). the putative orf encoded by the b . napus cdna is 96 amino - acids in length with a calculated molecular mass of 10 . 3 kda and that of the a . thaliana gene 107 amino - acids with a mass of 11 . 6 kda . although no overall homology was found to the putative a9 proteins by searching the nbrf protein database the a9 protein contains a cysteine motif that is present in several 2s plant storage proteins and in some plant protease inhibitors . the use of the a9 and a3 promoters to drive the expression of β - glucuronidase in anthers of arabidopsis thaliana and nicotiana tabacum . to demonstrate that the putative promoter regions of a3 and a9 are capable of driving the expression of a foreign gene in a . thaliana and n . tabacum , transcriptional fusions of the promoters were made to the escherichia coli gene encoding β - glucuronidase ( gus ). the 1030 bp hincii fragment of g3 . 6 is subcloned between into the hincii site of the vector ptz18 ( pharmacia ltd .) forming prs5 ( fig9 ). this is cut with ssti and the fragment cloned into ssti - cut pic19h ( marsh et al ., gene 32 481 - 485 ( 1984 )) forming pwp87 . the a3 promoter is then recovered as a hindiii - nrui fragment from pwp87 and cloned into hindiii , smai - cut pbi101 . 1 ( jefferson et al ., embo j 6 3901 ( 1987 )). the resultant plasmid ( pwp92 ) ( fig9 ) contains 745 bp of a3 sequence upstream of the ssti site ( position 745 bp in fig4 ) fused to gus . the 329 bp hincii - rsai fragment ( positions 1105 - 1434 bp in fig7 ) was cloned into hincii - cut ptz18 forming pwp70a . dna sequence analysis revealed the loss of a ` g ` residue at the rsai , hincii junction which resulted in the recreation of the rsai site . the hindiii , bamhi fragment of pwp70a containing the a9 promoter was cloned into bamhi , hindiii - cut pbluescript ( stratagene ) forming pwp71 . to reconstruct plasmids with larger a9 upstream regions the ecori , hindiii fragment of pwp71 was replaced with the 900 bp hindiii , ecori fragment of pwp64 ( fig6 ) ( which contains a 1486 bp acci , bglii fragment cloned into acci , bamhi - cut ptz19 ) forming pwp72 . also the ecori , hindiii fragment of pwp71 was replaced with the 1397 bp hindiii , ecori fragment of pwp55 ( fig6 ) ( which contains a 3146 bp xbai fragment cloned into xbai - cut ptz19 ) forming pwp73 . the hindiii , xbai fragments of pwp71 , pwp72 and pwp73 were cloned into hindiii , xbai - cut pbi101 . 1 forming pwp74 , pwp75 and pwp76 respectively . thus pwp74 contains a 329 bp a9 promoter fragment ( positions 1108 - 1437 bp ), pwp75 a 936 bp a9 fragment ( positions 501 - 1437 bp ) and pwp76 a 1437 bp a9 fragment ( positions 1 - 1437 bp ) all fused to gus . all the gus constructs are then transformed into n . tabacum and a . thaliana using standard transformation techniques . analysis of transformed plants demonstrated that gus activity was localised to anther tissues , specifically to tapetal cells . the temporal regulation of gus activity was identical to the temporal expression observed for the a3 and a9 genes as described in example 1 . fig1 shows the activity of the a9 - gus fusions in the anthers of transgenic tobacco plants . gus activity was assayed fluorometrically in the anthers staged precisely in terms of the development of the sporogenous cells . the pattern of expression of gus was the same ( quantitatively and qualitatively ) irrespective of the length of upstream region employed in the fusion . these experiments clearly demonstrate that the a9 promoter drives transcription in tapetal cells through a period commencing at the meiocyte stage of development and terminating during early microspore interphase . the construction of expression cassettes and their use in producing sense and anti - sense rna to anther - specific messages in transgenic plants . either anther - specific or constitutive promoters can be used to drive the expression of sense or anti - sense rna corresponding to anther - specific transcripts in transgenic plants , thus potentially creating anther mutations and male - sterility . the same anther - specific promoters can be used to drive the anther - specific expression of genes encoding proteins or enzymes detrimental to anther function thereby creating male - sterility . the use of the expression cassettes , the construction of which is described in this example , for this application are described in examples 4 and 5 . a ) construction of an intermediate vector to express sense and anti - sense rna utilising the a9 promoter . pwp72 ( fig1 ) is digested with xbai and religated , thus removing the bamhi site in the polylinker , forming pwp78 ( fig1 ). the kpni , ssti ( the ssti end rendered blunt with klenow ) a9 promoter fragment of pwp78 is ligated into kpni , smai - cut pjit60 forming pwp80 ( fig1 ). pjit60 is identical to pjit30 ( guerineau et al ., plant mol . biol . 15 , 127 - 136 ( 1990 )) except that the camv 35s promoter is replaced by a double camv 35s promoter ( guerineau et al ., nuc . acids res . 16 ( 23 ) 11380 ( 1988 )). the pwp80 intermediate vector consists of a 936 bp a9 promoter fragment fused to a polylinker derived from pbluescript with a 35s camv polyadenylation signal to stabilise the transcript . b ) construction of an intermediate vector to express sense and anti - sense rna utilising the a3 promoter . the camv promoter of pjit60 is replaced with the a3 promoter by cloning the 745 bp kpni , hindiii fragment of pwp87 ( fig9 ) into kpni , hindiii - cut pjit60 forming pwp88 ( fig1 ). pwp80 and pwp88 are therefore identical apart from the promoter region and surrounding restriction enzyme sites . c ) construction of chimeric genes containing the tapetum - specific a9 promoter linked to the sense or anti - sense orientation of the a9 cdna . anther - specific b . napus cdnas were cloned into ecori - cut lambda zapii by the addition of ecori linkers ( pharmacia ltd ) to the ends of the cdna . these linkers aim contain internal noti sites , so the entire cdna can be recovered as a noti fragment provided the cdna contains no internal noti sites . the b . napus cdna for a9 is therefore recovered as a noti fragment and cloned in both orientations ( sense and anti - sense ) into noti - cut pwp80 . the promoter , cdna and terminator regions are excised from the pwp80 derivatives with a hindiii , xhoi digest and are cloned into sali , hindiii - cut pbin19 ( bevan et al ., nuc . acids res . 22 8711 - 8721 ( 1984 )). the pbin19 derivatives are transformed into b . napus . the resulting transgenic plants expressing anti - sense a9 rna are male sterile . other chimeric genes that can be constructed to produce male sterility are : i ) a9 promoter linked to the coding region of the a . thaliana a9 gene , such that anti - sense a9 rna is expressed ; ii ) a3 promoter driving expression of anti - sense a9 , either from the a9 cdna or from the a . thaliana a9 gene ; iii ) a9 promoter expressing anti - sense rna to a3 , using either the a3 cdna or the a . thaliana a3 gene ; iv ) a3 promoter expressing anti - sense rna to a3 , using either the a3 cdna or the a . thaliana a3 gene . these plasmids could also be transformed into other members of the brassicaceae causing male sterility in the transgenic plants . construction of chimeric a3 - barnase and a9 - barnase genes and their expression in transgenic plants . to demonstrate the utility of the a3 and a9 promoters they are used to drive the expression of the rnase , barnase , in tapetal cells . use of the barnase gene to create male sterile plants has been described in ep - a - 0344029 ( plant genetic systems ) and has been published by mariani et al ., nature 347 , 737 - 741 . the oligonucleotide primers seq id no : 13 ! and seq id no : 14 ! : ## str1 ## are used in a polymerase chain reaction ( pcr ) to generate a fragment encoding barstar and the mature barnase product from the plasmid ptg2 ( horovitz et al ., j . mol . biol . 216 , 1031 - 1044 ( 1990 )). the first primer is homologous to nucleotides 95 - 221 bp of fig1 in hartley r . w . j . mol . biol . 202 , 913 - 915 ( 1988 ). the second primer is homologous to a sequence immediately next to the hindiii site of ptz18u ( pharmacia ). barstar is retained on this fragment since active barnase cannot be cloned in the absence of the specific inhibitor barstar ( only barnase is expressed in the transgenic plants ). the pcr fragment is digested with xbai , hindiii and cloned into xbai , hindiii - cut pbluescript forming pwp120 ( fig1 a ). pwp120 is digested with xbai , hincii and the barnase / barstar fragment cloned into xbai - smai cut pwp91 forming pwp127 in which the a9 promoter is transcriptionally fused to the mature barnase sequence ( fig1 b ) ( pwp91 is identical to pwp80 except that the polylinker region between xbai and ecori has been replaced with the sites spei , bamhi , smai and psti ). this gene fusion is transferred to pbin19 by ligating the xhoi fragment - of pwp127 to sa / i - cut pbin19 . b ) translational fusion of the a9 promoter and gene to barnase the primers seq id no : 15 ! and seq id no : 16 ! ## str2 ## are used in a pcr reaction to generate an a9 promoter fragment from pwp64 ( fig6 ) that contains the entire 5 &# 39 ; untranslated region of the a9 gene and has the sequence around the initiating methionine of the a9 gene mutated to an ncoi site ( the second primer is homologous to a sequence within the ptz19u vector ). this fragment is cut with hindiii , xbai and cloned into pwp80 or pwp91 replacing the existing a9 promoter fragments in these intermediate vectors . the new intermediate vectors are pwp112 and pwp113 respectively . pwp120 is cut with ncoi , hincii and the barnase / barstar fragment cloned into ncoi , smai - cut pwp113 forming pwp128 ( fig1 b ). the chimeric gene is then cloned as an xhoi fragment into the sali site of pbin19 . the a9 promoter of pwp127 is replaced with the a3 promoter by cutting pwp127 with sali , xbai and cloning in the sali , xbai a3 promoter fragment of pwp88 forming pwp131 ( fig1 b ). the chimeric gene is transferred as a kpni , xhoi fragment into kpni , sali - cut pbin19 . the pbin19 derivative plasmids are transformed into n . tabacum where expression of barnase in transgenic plants results in the degradation of the tapetal cells of the anther causing complete male sterility . the plants are female fertile . thus both the a3 and a9 promoters are tapetum - specific and are suitable for driving the expression of any cytotoxic agent within tapetal cells leading to the production of male sterile , but otherwise phenotypically normal transgenic plants . these plasmids could also be transformed into other crop species such as b . napus , zea mays and hordeum vulgare leading to male sterility in the transgenic plants . construction and expression in transgenic plants of chimeric gene fusions between the a3 and a9 promoters and a β - 1 , 3 glucanase gene . the temporal pattern of expression of the a3 and a9 genes determined from northern analysis ( example 1 ) and promoter - gus fusions ( example 2 ) show that both promoters are active at stages of anther development prior to the release of microspores from tetrads . thus either promoter is suitable for driving the premature expression of β - 1 , 3 glucanase in anthers leading to male sterility , as previously discussed . cdnas encoding an n . tabacum basic β - 1 , 3 glucanase has been described by shinshi et al . ( pnas 85 5541 - 5545 ( 1988 )) and neale et al . ( plant cell 2 673 - 684 ( 1990 )). this enzyme is located in the vacuole and it has been suggested that a c - terminal sequence may be responsible for its intracellular location ( van den bulcke et al ., pnas 86 2673 - 2677 ( 1989 )). an engineered cdna for this glucanase is cloned by using two oligonucleotides complementary to the sequence of an n . tabacum glucanase ( shinshi et al ., 1988 , neale et al ., 1990 ) and using the polymerase chain reaction to isolate a glucanase cdna from n . tabacum mrna . the first oligonucleotide has sequence seq id no : 17 ! : this primer contains an xbai and an ncoi site followed by a sequence identical to a 5 &# 39 ; region of glucanase ( positions 7 - 29 in shinshi et al ., 1989 ). the second oligonucleotide has sequence seq id no : 18 ! : this primer has a sacii site followed by a trinucleotide that is a stop codon and a sequence complementary to the region that encodes the c - terminus of the mature glucanase ( positions 1017 - 993 in shinshi et al ., 1988 ). the glucanase is therefore cloned with restriction enzyme sites at both ends for ligation into the intermediate vectors pwp80 and pwp88 . also the enzyme is been engineered so that the c - terminal targeting signal is removed . the enzyme will therefore be secreted rather than be directed to the vacuole when expressed in transgenic plants . the glucanase gene is cloned as an xbai , sacii fragment into xbai , sacii - cut pwp80 forming a transcriptional fusion between the a9 promoter and glucanase forming pdw80pr . an a3 - glucanase transcriptional fusion is constructed by replacing the sali , xbai a9 promoter region of pdw80pr with the sali , xbai a3 promoter fragment of pwp88 forming and pdw88pr ( fig1 a ). a translational fusion of the a9 promoter and gene to the glucanase is made by cloning the glucanase as an ncoi , sacii into ncoi , sacii - cut pwp112 forming pdw112pr ( fig1 b ). the chimeric genes in pdw80pr and pdw112pr are transferred as saci , ecorv fragments into sali , smai - cut pbin19 and the chimeric gene in pdw88pr transferred as a sali , ecorv fragment into sali , smai - cut pbin19 . the pbin19 derivatives are transformed into n . tabacum . callose surrounding the microsporocytes prematurely disappears in the transgenic plants causing male sterility . these plasmids could also be transformed into other crop species such as b . napus , zea mays and hordeum vulgare leading to male sterility in the transgenic plants . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 18 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 347 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 3 .. 329 ( d ) other information : / codon_start = 3 / product =&# 34 ; bn a3 gene &# 34 ;( xi ) sequence description : seq id no : 1 : tttcttctttctgtttactcctcctcgtcgtcttcttcctcaattct47serserphecysleuleuleuleuvalvalphepheleuasnser151015caacctgcactctcactccgtgtcccaaaaccgcagtcagaaccagca95glnproalaleuserleuargvalprolysproglnsergluproala202530tcaccacaaaccatgatcgatgactcatctccaatgggaatgatcgac143serproglnthrmetileaspaspserserprometglymetileasp354045catgcaaagtccatgattgctggattcttcagccacaagtttccagta191hisalalyssermetilealaglyphepheserhislyspheproval505560atgggctggcctttccccaagtacccacctttcacaatggtcaaccct239metglytrppropheprolystyrproprophethrmetvalasnpro657075aacgttccaacaaacccatctggagctcaagaggaatcagagaagcta287asnvalprothrasnproserglyalaglnglugluserglulysleu80859095ccttcttccccaagcaaacttaacaaagctggacgaaacgca329proserserproserlysleuasnlysalaglyargasnala100105tgaaaattgttgttggaa347 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 109 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; bn a3 amino acid &# 34 ;( xi ) sequence description : seq id no : 2 : serserphecysleuleuleuleuvalvalphepheleuasnsergln151015proalaleuserleuargvalprolysproglnsergluproalaser202530proglnthrmetileaspaspserserprometglymetileasphis354045alalyssermetilealaglyphepheserhislyspheprovalmet505560glytrppropheprolystyrproprophethrmetvalasnproasn65707580valprothrasnproserglyalaglnglugluserglulysleupro859095serserproserlysleuasnlysalaglyargasnala100105 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 422 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 2 .. 334 ( d ) other information : / codon_start = 2 / product =&# 34 ; bn e5 gene &# 34 ;( xi ) sequence description : seq id no : 3 : aaaggtttcttctttctgtttactcctcctcgtcgtcttcttcctc46lysvalserserphecysleuleuleuleuvalvalphepheleu151015aattctcaacctgcactctcactccgtgtcccaaaaccgcagtcagaa94asnserglnproalaleuserleuargvalprolysproglnserglu202530ccagcatcaccacaaaccatgatcgatgactcatctccaatgggaatg142proalaserproglnthrmetileaspaspserserprometglymet354045atcgaccatgcaaagtccatgattgctggattcttcagccacaagttt190ileasphisalalyssermetilealaglyphepheserhislysphe505560ccagtaatgggctggcctttccccaagtacccacctttcacaatggtc238provalmetglytrppropheprolystyrproprophethrmetval657075aaccctaacgttccaacaaacccatctggagctcaagaggaatcagag286asnproasnvalprothrasnproserglyalaglnglugluserglu80859095aagctaccttcttccccaagcaaacttaacaaagctggacgaaacgca334lysleuproserserproserlysleuasnlysalaglyargasnala100105110tgaaaattgttgttggaaacatttctatggttttacattttcattaaaataaaataaaat394tgtatctttaacaattgaatggtaagcg422 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 111 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; bn e5 amino acid &# 34 ;( xi ) sequence description : seq id no : 4 : lysvalserserphecysleuleuleuleuvalvalphepheleuasn151015serglnproalaleuserleuargvalprolysproglnserglupro202530alaserproglnthrmetileaspaspserserprometglymetile354045asphisalalyssermetilealaglyphepheserhislysphepro505560valmetglytrppropheprolystyrproprophethrmetvalasn65707580proasnvalprothrasnproserglyalaglnglugluserglulys859095leuproserserproserlysleuasnlysalaglyargasnala100105110 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 398 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 1 .. 315 ( d ) other information : / codon_start = 1 / product =&# 34 ; bn e3 &# 34 ;( xi ) sequence description : seq id no : 5 : gccctcctcgtcgtcgtcttcttcctcagttctcaacctgcactctca48alaleuleuvalvalvalphepheleuserserglnproalaleuser151015ctccgtctcccaaaaccgcagtcagaactagcatcacctcaaaccatg96leuargleuprolysproglnsergluleualaserproglnthrmet202530atcgatgactcatctccaatgggaatgatcgaccatgcaaaagccatg144ileaspaspserserprometglymetileasphisalalysalamet354045attgctggattcttcagccacaagtttccactaatgggctggcctttc192ilealaglyphepheserhislyspheproleumetglytrpprophe505560ccaaagtacccacccttcacaatggttaaccctaacgttccaacaaaa240prolystyrproprophethrmetvalasnproasnvalprothrlys65707580ccatctggagctcaagaggaatcagagaagttaccttcttccccaagc288proserglyalaglnglugluserglulysleuproserserproser859095aaacttaacaaagatggacgaaacgcatgaaaattgctgttggaaac335lysleuasnlysaspglyargasnala100105atttctatggttttacattttcattaaaataaagaaattgtatctttaacaattgaatgg395taa398 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 105 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; bn e3 amino acid &# 34 ;( xi ) sequence description : seq id no : 6 : alaleuleuvalvalvalphepheleuserserglnproalaleuser151015leuargleuprolysproglnsergluleualaserproglnthrmet202530ileaspaspserserprometglymetileasphisalalysalamet354045ilealaglyphepheserhislyspheproleumetglytrpprophe505560prolystyrproprophethrmetvalasnproasnvalprothrlys65707580proserglyalaglnglugluserglulysleuproserserproser859095lysleuasnlysaspglyargasnala100105 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 1276 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 770 .. 1126 ( d ) other information : / codon_start = 770 / product =&# 34 ; at a3 gene &# 34 ;( xi ) sequence description : seq id no : 7 : gtcgacactatttgttaatcagtagactcatttgtgctgcctttggcttcttttcgtcgg60aaaaacagctgaatgtgactctaatttcggcggcagcaaacgcggcggcgttcatggcgg120aggtaggaaagaacgggaacaaacacgcgagatgggacaaaatctgtgacagattcgcca180cttactgtgatcacggcgccggagcattaatcgccgcattcgccggcgtaatccttatgc240tcatcatctccgccgcgtcaatctctcgtctcgtccaacctaataaatgctgctccacca300ccgcatctccctcggtcgtcccctgaaaacatacacacgtgtcacgttcccctgttattt360tccttttcttttcttacttctttgtttcttcttgagtgtgtggtgtgtatctcgagtgaa420tgtgattgatcagattgtgtaagcatttggctcgtctcttctttgtaaaactttcttctt480atggtttattcttaaaccaattctccgactagggttaggttactaaaatatccattacct540atactcgttatcaatacccatactcgttatatcaaccataaatcatctctcatagtcatg600ctcaaatcatgaacctttttaattcgaatactggtttaggtaagaagtagaatcccaacg660taaaaaacacatctcttctacaaactcaaaaatcactactatataagattaccaagaaac720cccataaaacacaatcacaaacaagagctcaaaaacaccaaagcaaacaatgtcg775metseraaaatctcaaaagcttcttctctctgtttactccttctcgtcttcttc823lysileserlysalaserserleucysleuleuleuleuvalphephe51015ctcttcagttcccgacccgcactctcactccgtggcccaaaacttcaa871leupheserserargproalaleuserleuargglyprolysleugln202530tcatcggaaccaaaatcagctcaaaccctaatggatgattcgtcttca919sersergluprolysseralaglnthrleumetaspaspserserser35404550atgaacaagatcaactctggaaatgcaaaaaccatgattgctggtttc967metasnlysileasnserglyasnalalysthrmetilealaglyphe556065ttcagtcacaagtttccattaaagggctggcctttccctaagtaccca1015pheserhislyspheproleulysglytrppropheprolystyrpro707580cctttccctatggttaaccctaatattgcaacaaacccatctggagct1063propheprometvalasnproasnilealathrasnproserglyala859095caagaggaatccgcaaagttaccttcttctccaagcaaagacaacaaa1111glnglugluseralalysleuproserserproserlysaspasnlys100105110gatggacgaaacgcttgaagattagagtttacttatttatatggtttttaccatt1166aspglyargasnala115gcatcaaataaaaaatgtacctttaacaattaaatggtaaaagaaaaaagatatttatat1226atcatcagctcgaagcgattttgttatgacagttacaggaatattaacaa1276 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 119 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; at a3 amino acid &# 34 ;( xi ) sequence description : seq id no : 8 : metserlysileserlysalaserserleucysleuleuleuleuval151015phepheleupheserserargproalaleuserleuargglyprolys202530leuglnsersergluprolysseralaglnthrleumetaspaspser354045sersermetasnlysileasnserglyasnalalysthrmetileala505560glyphepheserhislyspheproleulysglytrppropheprolys65707580tyrpropropheprometvalasnproasnilealathrasnproser859095glyalaglnglugluseralalysleuproserserproserlysasp100105110asnlysaspglyargasnala115 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 490 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 9 .. 296 ( d ) other information : / codon_start = 9 / product =&# 34 ; bn a9 gene &# 34 ;( xi ) sequence description : seq id no : 9 : taaacaaaatggaatttctcaaatcctttacaactattctctttgtaatg50metglupheleulysserphethrthrileleuphevalmet1510tttctggccatgagcgctctggagaccgtacctatggttcgagctcaa98pheleualametseralaleugluthrvalprometvalargalagln15202530caatgcctagacaatttgagcaatatgcaggtgtgtgcgccgctggtt146glncysleuaspasnleuserasnmetglnvalcysalaproleuval354045ctgcctggtgcagtcaatccagccccgaattcaaattgctgcattgct194leuproglyalavalasnproalaproasnserasncyscysileala505560ctccaagcaactaacaaagattgtatatgtaacgcccttcgagcagcc242leuglnalathrasnlysaspcysilecysasnalaleuargalaala657075accacatttaccactacttgcaacctcccctctttagattgtggtata290thrthrphethrthrthrcysasnleuproserleuaspcysglyile808590accatatgagtggtttcagcaacggtcagttccgaggatttggggagtttggtctg346thrile95caaaagacaacaagaataaaagtattaaaataacgagaaagtgtgtgtgtttttttttaa406tttggtctgttgttcggttggttcaatacttaaatatgacccatcaattaatatcgtttt466catatttattatggtaatattttt490 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 96 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; at a9 amino acid &# 34 ;( xi ) sequence description : seq id no : 10 : metglupheleulysserphethrthrileleuphevalmetpheleu151015alametseralaleugluthrvalprometvalargalaglnglncys202530leuaspasnleuserasnmetglnvalcysalaproleuvalleupro354045glyalavalasnproalaproasnserasncyscysilealaleugln505560alathrasnlysaspcysilecysasnalaleuargalaalathrthr65707580phethrthrthrcysasnleuproserleuaspcysglyilethrile859095 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 1952 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 1461 .. 1781 ( d ) other information : / codon_start = 1461 / product =&# 34 ; at a9 gene &# 34 ;( xi ) sequence description : seq id no : 11 : tctagacataacggtgagagttaatattaaaatttcaggcgagaaaaatgatacttgaaa60aatattatgatcgttttggatattccttacatcgagtgaatgttggtttgattcatcttc120caagtgttctgcaaacgtatattaaaggtttattaactggtaagagattaaccgggtttt180ggttcagcatataccatgattgactaactgatcaaatagtctttacttattatataaaga240cgatactattggtcatgctacaaaatcaagtcataccatatcctgagaatgaatgtggag300aatcgttataaggcataagtgtgggtattgatcgtggtacgaacaaccgccttggcatca360acattagccacgatatccaacatttgaagcattgcctatggcgagtgtttggttggtttt420gaaactgatgatgataaccagaacgagaaatgtcttgtgaagtataatgttccgatgaat480tgggattataataatgtgtagacattgtaggttggttttgatgatgataagtaatcattg540gagaattgtctaacacatgcactggagaattattgactctaccacgttctctttgatatt600cctcgattttcctcgtgatttcatcagcctctccgaaaaagtaattgtatccactagaac660tttgggaatctcccatctaatttatgtattagagaagttataatattttggggaaataga720ttttctctactgattttgttgtgtgacattatatttttataagtacatgtttctgtttcg780ttatattgttgtcgtggttgagtctttattagagcatgtaaatatgtttatgaaataagc840gagaaaggaattaattaaacgtatcgagtgataaatgctttaatggattcgagatttagt900attcttaaatttttgtttcattatcattgattataaaactaagttatgttgatctcaaat960ccttaattatgttctcctaagaagagtacaagtggtgggaacgaaagatgagtaaaatac1020taaaaatcttttctcaaaagtcaaatcgcattagttaacaaaaacaaaccatgtgttacc1080gtcaaatcaatgtgtttaaaagatgttaaccactaatcaagcatttacgtgtaaccggat1140caaccggatttgggttttgaatatgttgtggagatgtatataaatgataaattaattgaa1200tatcttaattaatctgtgaaagaaactacatcacacactttgttatttcccctagctttt1260agtttttttatcatgcaaaacttatgaagtaactagatcaagatcacaaaaaaaaagcat1320cacttcacttcatgacctaattattctcgaagcccaaaactatttacatacacttttatt1380ctataaatatagatgatggaattcaccaatccaaaagtgaataaaaaacacaagtacaaa1440caatatagtatctaattagaatggtatctctaaagtcccttgctgctatt1490metvalserleulysserleualaalaile1510ctcgttgccatgtttcttgccaccggacctacggttctagcccagcag1538leuvalalametpheleualathrglyprothrvalleualaglngln152025tgcagagacgaactgagcaatgtgcaggtgtgcgcgccgctgcttctg1586cysargaspgluleuserasnvalglnvalcysalaproleuleuleu303540cccggtgcggtcaatcctgccgcgaactcaaattgctgcgctgccctc1634proglyalavalasnproalaalaasnserasncyscysalaalaleu455055caagcaactaacaaagattgtctatgtaaccgtcttcgagcagccacc1682glnalathrasnlysaspcysleucysasnargleuargalaalathr606570acacttacctctctttgtaacctcccctcttttgattgtggtaagatg1730thrleuthrserleucysasnleuproserpheaspcysglylysmet75808590atccatcgattaaaaccttttttactagatttttataaattattccat1778ilehisargleulyspropheleuleuaspphetyrlysleuphehis95100105caatagtgtttgttttatatttgttctcatgattttttggacttatgttttgt1831glngaactgtgcaggcataagtgcctagttgaacaacattcagttccgaggatttggggagtt1891tggtctgcaaacgacaagacgaataaaataaaataatgagaaatacactatttagtgttt1951t1952 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 107 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( ix ) feature :( d ) other information : product =&# 34 ; at a9 amino acid &# 34 ;( xi ) sequence description : seq id no : 12 : metvalserleulysserleualaalaileleuvalalametpheleu151015alathrglyprothrvalleualaglnglncysargaspgluleuser202530asnvalglnvalcysalaproleuleuleuproglyalavalasnpro354045alaalaasnserasncyscysalaalaleuglnalathrasnlysasp505560cysleucysasnargleuargalaalathrthrleuthrserleucys65707580asnleuproserpheaspcysglylysmetilehisargleulyspro859095pheleuleuaspphetyrlysleuphehisgln100105 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 13 : gggtctagaccatggcacaggttatcaacacgtttgacgg40 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 14 : gtaaaacgacggccagtgcc20 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 40 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 15 : gggtctagaccatggtaattagatactatattgtttgtac40 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 18 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 16 : aatacgactcactatagg18 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 34 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 17 : cgctctagaccatggctgctatcacactcctagg34 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 37 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 18 : gggccgcggtcacccaaagttgatattatatttgggc37__________________________________________________________________________