Patent Application: US-13627793-A

Abstract:
the cdna and genomic dna sequences encoding the human adrenoleukodystrophy protein are provided . mutations of the adrenoleukodystrophy protein cause adrenoleukodystrophy or adrenomyelopathy .

Description:
here a positional cloning was used to identify a gene partially deleted in 6 of 85 independent patients with ald . in familial cases , the deletions segregated with the disease . an identical deletion was detected in two brothers presenting with different clinical ald phenotypes . candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary dnas by exert connection and screening of cdna libraries . the deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein or m r 70k that is involved in peroxisome biogenesis and belongs to the &# 34 ; atp - binding cassette &# 34 ; superfamily of transporters . as previous attempts to purify vlcf - coa synthetase were unsucessful , a &# 34 ; positional cloning &# 34 ; approach was used . the ald locus has been mapped to xq28 ( deduve , c . et al , biochem . pharmac . 23 , 2495 - 2531 , 1974 ), where the red / green colour pigment ( r / gcp ) genes reside . on the basis of the high incidence ( 40 %) of colour vision anomalies in amn patients and earlier results , it was firstly proposed that ald and r / gcp genes could be close together . recently , an amn patient with blue - monochromatic colour vision was identified who had a complex rearrangement located 5 &# 39 ; of the red - colour pigment ( rcp ) gene , which included two deletions separated by a large (& gt ; 110 kb ) inversion ( fig1 a and b ). only the rcp gene was found in the first deletion ( 88 kb ). no additional deletion was detected in this region in 81 other ald patients . it was then postulated that the inverted segment or the second deletion were candidate loci for the ald gene . probes cot - responding to three of the breakpoints ( bp ) of this rearrangement were isolated ( bp1 , bp2 and bp3 ) ( fig1 ). to estimate the size of the second deletion a probe 4 kb proximal to fr15 was used ( deleted in patient o ; martinez et al , cell biol . int . rep . 14 , 255 - 266 ( 1990 ) to obtain clones from a xq28 cosmid library . three overlapping clones were obtained that spanned about 90 kb and contained a cluster of rare restriction cutting sites ( eagi , bsshii , sacii ) indicating the presence of a cpg island ( fig1 c ). in parallel , an xbai junction fragment ( x - 8 ) corresponding to breakpoints 4 and 2 in patient o was cloned ( fig1 a ) . a restriction map of x - 8 showed that a 1 - 8 kb xbai - ecori ( x2 ) fragment contains a 1 . 6 - kb segment on the bp4 side and included within the fr15 cosmid contig described above ( fig1 b and c ). breakpoints 3 and 4 are separated by 19 . 2 kb , and delimit the second deletion in patient o . to search for conserved sequences in other mammalian species and to detect additional deletions in other ald patients , cosmid qc 11h12 was subcloned ( fig1 b , c ). probes ta25 , ta1 , ta18 and ta13b ( fig1 c ) showed cross - hybridization to various mammalian species . more important , x2 , ta4 , ta18 and ta13a probes detected deletions in five other ald patients . probe x2 allowed the detection of a junction fragment segregating with the disease in family b ( fig1 d ). in another family ( ma ), probe x2 detected an identical 22 - kh hindiii junction fragment in two brothers with different clinical phenotypes of ald ( fig1 e ). the size of deletions ranged from 1 . 6 kb ( patient b ) to 15 . 3 kb ( patient r ) with partial overlap ( fig1 c ). six patients had deletions ( including patient o ) in a population of 85 independent ald patients , but no deletions were found in a panel of 82 control males . these results indicate that the region contains at least part or the ald gene . the sequences of probes ta25 , ta18 , ta1 . 3b and of ta13a were determined and examined for putative coding regions ( using a computer program based on a multiple sensor - neural netword approach ) revealed a large (& gt ; 700 bp ) putative protein coding sequence within ta25 and smaller open reading frames ( 300 - 400 bp ) in ta18 , ta13 a and ta13b . the deduced amino - acid sequences showed significant sequence identity with collinearly positioned regions of human or rat 70k peroxisomal membrane protein ( pmp ). nested - pcr reactions using primers from the putative exons ( fig2 ) produced two fragments ( ex13 and ex3 ) of sizes ( 645 bp and 498 bp , respectively ) compatible with those expected from homology with the 70k pmp cdna . they hybridized to the predicted dna fragments in normal individuals and to the same fragments detected by genomic probes in ald patients ( fig3 a ). ex13 and ex3 have been used in combination to screen a random - primed hela cell cdna library to obtain 6 independent overlapping clones . the 2 , 751 - bp sequence ( fig2 ) ( seq id no : 2 ) contains the whole protein - coding sequence of 745 amino acids ( seq id no : 2 ). the first methionine codon is preceded by an in - phase stop codon ( at bp 282 ) and is included within a potential ribosome - binding sequence . significant sequence identity with the 70k pmp ( seq id no : 3 ) begins at methionine 67 and ends at around 680 ( corresponding to the carboxy terminus of 70k pmp ). the remaining 52 amino acid residues are unique to the ald protein ( fig4 a ). the sequence of ald protein ( seq id no : 2 ) could be aligned with human 70k pmp ( seq id no : 3 ) ( saari , j . c . & amp ; bredberg , l . biochim . biophys . acta 716 , 266 - 272 , 1982 ) ) with only a few deletions or insertions , and revealed a 38 . 5 % amino - acid identity ( 253 / 659 amino acids ) ( fig4 a ). when conservative amino acids substitutions are considered , the sequence similarity between the two protein sequences reached 78 . 9 %. the two proteins show a similar hydrophobicity profiles , with a hydrophobic amino - terminal region containing potential transmembrane segments . the hydrophilic carboxy - terminal region of the ald protein shows 56 % identity over 210 amino acids to the corresponding region of 70k pmp . the two characteristic nucleotide - binding consensus sequences are almost identical between the two proteins ( underlined in fig4 b ). when northern blots of poly ( a ) + rnas from human tissues were hybridized to probes ex13 or ex3 , a transcript of 4 , 2 kb was detected in heart , placenta , lung , liver , skeletal muscle , testis , pancreas and , to a lesser extent , in brain and kidney ( fig3 b ). the expression of the 4 . 2 - kb transcript was very low in adult brain but more marked in 21 - week fetal brain . a second transcript of 6 . 8 kb was detected in heart and skeletal muscle , whereas a third transcript of 2 . 75 kb was detected in muscle and liver . these additional transcripts could arise from alternative processing or the use of multiple polyadenylation sites . the sequence shown in fig2 corresponds to 4 . 2 - kb messenger rna , this being the only species detected in hela cells . the putative ald gene has thus been identified in the distal part of xq28 which has deletions in one or several exons in 6 of 85 independent ald patients . some of these deletions are small and non - overlapping , thus strengthening the conclusion that this gene is indeed involved in ald . although the gene coding for vlcfa - coa synthetase was considered as a candidate gene for ald , a recently cloned rat gene for long - chain ( c 12 - c 16 ) acyl - coa synthetase failed to detect homologous sequences on the x chromosome . the putative ald gene shows no homology to this latter sequence , or to the three other enzymes involved in peroxisomal β - oxidation . surprisingly , a very significant sequence identity was found with human and rat 70k pmp . two putative domains could be identified by hydropathy analysis : an amino - terminal hydrophobic region , which presumably contains six transmembrane segments , and a hydrophilic region containing atp - binding motifs with striking identity to the atp - binding region of the human 70k pmp . this sequence is well conserved in the atp - binding cassette ( abc ) family of transporters , which include the multidrug - resistant gene product , the cystic fibrosis transmembrane conductance regulator , and the products of the psf1 and psf2 genes , which encode peptide transporters and map in the class ii region of the human mhc complex ( fig4 b ). the conserved region includes the a and b consensus sequences ( a ; g - x4 - g - k - t - x6 - i / v ; b : r / k - x3 - g - x3 - l ( hydrophobic ) 4 - d ) of a nucleotide - binding fold , plus a 12 - amino - acid motif highly conserved in abc proteins ( fig4 b ). ald protein must therefore be a member of this superfamily of abc transporters , which are also involved in transport of proteins , amino acids , inorganic ions and peptides in prokaryotes and eukaryotes . although the predicted sequence of ald protein ( seq id no : 2 ) shows significant identity to 70k pmp ( seq id no : 3 ), no homology was found to 35k pmp or to other pmps required for peroxisome biogenesis in yeast . although ald was initially thought to involve a deficiency in peroxisomal vlcfa - coa synthesase , the predicted sequence of the putative ald protein rather suggests a protein involved in transport of vlcfa - coa synthetase into the peroxisomal membrane or a protein that is functionally associated with the vlcfa - coa synthetase in the peroxisomal membrane . the translocation of acyl - coa oxidase , the next enzyme or the peroxisomal β - oxidation pathway , requires atp hydrolysis , whereas the transport of vlcfa across the peroxisomal membrane does not , and neither is it impaired in peroxisomes from ald patients . expression of ald protein was observed in every tissue tested , but the relationship between ald protein expression and the abundance of peroxisomes in tissues may not be straight - forward . perosisomes are particularly abundant in liver and kidney , having an average diameter of 0 . 2 - 1 μm . in other tissues , including the brain and fibroblasts , they are less abundant and smaller ( 0 . 05 - 0 . 2 μm ). this abundance and size difference may reflect a distinct membrane and matrix protein compositions of peroxisomes in different tissues . although ald is associated with a defective oxidation of vlcfa , this metabolic defect is mainly expressed in brain and adrenal tissues . ald is characterized by a striking variation in clinical phenotype . in family ma , an identical deletion was found in two sibs , a boy who developed cerebral ald at 8 years , and his brother who developed only very mild adrenal insufficiency at 13 years . furthermore , deletions were associated with the adult form ( patients o and r ) as well as with the severe childhood form ( patients b and l ). these differences suggest that the phenotypic variability of ald is probably due to secondary factors ( possibly immunological ) or to the influence of still unidentified modifier genes . the defective vector still contains its cis - sequences such as the ltr sequence or part the thereof sufficient for the transcription and integration , the psi sequence necessary for the encapsidation and the pb sequence for vital replication . in this vector the viral genes gag , pol , env are at least partly deleted . the promoter of the ald gene or advantageously a stronger promoter such as the pgk - 1 promoter ( phosphoglycerate kinase ) or sv10 promoter replace the deleted vital gene . the cdna of the ald gene is cloned within the defective vector , under the control of the promoter , in a chosen restriction site . the retroviral vector is then introduced in a cell line for encapsidation , which cell line expresses the gag , pol and env vital genes . a cell line like nih / 3t3 previously modified with the helper virus ( danos et al , pnas , 85 , 6460 - 6465 , 1988 ) is used . the recombinant construct is introduced by transfection and the cells produce vital particles . the cells used for the transfer of the cdna sequence are cultivated and contacted and incubated with the retroviral vector . the infected cells are then amplified sufficiently to be used for the treatment . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 23 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 2750 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 387 .. 2624 ( xi ) sequence description : seq id no : 1 : gcggacggacgcgcctggtgccccggggaggggcgccaccgggggaggaggaggaggaga60aggtggagaggaagagacgccccctctgcccgagacctctcaaggccctgacctcagggg120ccagggcactgacaggacaggagagccaagttcctccacttgggctgcccgaagaggccg180cgaccctggagggccctgagcccaccgcaccaggggccccagcaccaccccgggggccta240aagcgacagtctcaggggccatcgcaaggtttccagttgcctagacaacaggcccagggt300cagagcaacaatccttccagccacctgcctcaactgctgccccaggcaccagccccagtc360cctacgcggcagccagcccaggtgacatgccggtgctctccaggccccggccc413metprovalleuserargproargpro15tggcgggggaacacgctgaagcgcacggccgtgctcctggccctcgcg461trpargglyasnthrleulysargthralavalleuleualaleuala10152025gcctatggagcccacaaagtctaccccttggtgcgccagtgcctggcc509alatyrglyalahislysvaltyrproleuvalargglncysleuala303540ccggccaggggtcttcaggcgcccgccggggagcccacgcaggaggcc557proalaargglyleuglnalaproalaglygluprothrglngluala455055tccggggtcgcggcggccaaagctggcatgaaccgggtattcctgcag605serglyvalalaalaalalysalaglymetasnargvalpheleugln606570cggctcctgtggctcctgcggctgctgttcccccgggtcctgtgccgg653argleuleutrpleuleuargleuleupheproargvalleucysarg758085gagacggggctgctggccctgcactcggccgccttggtgagccgcacc701gluthrglyleuleualaleuhisseralaalaleuvalserargthr9095100105ttcctgtcggtgtatgtggcccgcctggacggaaggctggcccgctgc749pheleuservaltyrvalalaargleuaspglyargleualaargcys110115120atcgcccgcaaggacccgcgggcttttggctggcagctgctgcagtgg797ilealaarglysaspproargalapheglytrpglnleuleuglntrp125130135ctcctcatcgccctccctgctaccttcgtcaacagtgccatccgttac845leuleuilealaleuproalathrphevalasnseralaileargtyr140145150ctggagggccaactggccctgtcgttccgcagccgtctggtggcccac893leugluglyglnleualaleuserpheargserargleuvalalahis155160165gcctaccgcctctacttctcccagcagacctactaccgggtcagcaac941alatyrargleutyrpheserglnglnthrtyrtyrargvalserasn170175180185atggacgggcggcttcgcaaccctgaccagtctctgacggaggacgtg989metaspglyargleuargasnproaspglnserleuthrgluaspval190195200gtggcctttgcggcctctgtggcccacctctactccaacctgaccaag1037valalaphealaalaservalalahisleutyrserasnleuthrlys205210215ccactcctggacgtggctgtgacttcctacaccctgcttcgggcggcc1085proleuleuaspvalalavalthrsertyrthrleuleuargalaala220225230cgctcccgtggagccggcacagcctggccctcggccatcgccggcctc1133argserargglyalaglythralatrpproseralailealaglyleu235240245gtggtgttcctcacggccaacgtgctgcgggccttctcgcccaagttc1181valvalpheleuthralaasnvalleuargalapheserprolysphe250255260265ggggagctggtggcagaggaggcgcggcggaagggggagctgcgctac1229glygluleuvalalagluglualaargarglysglygluleuargtyr270275280atgcactcgcgtgtggtggccaactcggaggagatcgccttctatggg1277methisserargvalvalalaasnserglugluilealaphetyrgly285290295ggccatgaggtggagctggccctgctacagcgctcctaccaggacctg1325glyhisgluvalgluleualaleuleuglnargsertyrglnaspleu300305310gcctcgcagatcaacctcatccttctggaacgcctgtggtatgttatg1373alaserglnileasnleuileleuleugluargleutrptyrvalmet315320325ctggagcagttcctcatgaagtatgtgtggagcgcctcgggcctgctc1421leugluglnpheleumetlystyrvaltrpseralaserglyleuleu330335340345atggtggctgtccccatcatcactgccactggctactcagagtcagat1469metvalalavalproileilethralathrglytyrsergluserasp350355360gcagaggccgtgaagaaggcagccttggaaaagaaggaggaggagctg1517alaglualavallyslysalaalaleuglulyslysgluglugluleu365370375gtgagcgagcgcacagaagccttcactattgcccgcaacctcctgaca1565valsergluargthrglualaphethrilealaargasnleuleuthr380385390gcggctgcagatgccattgagcggatcatgtcgtcgtacaaggaggtg1613alaalaalaaspalailegluargilemetsersertyrlysgluval395400405acggagctggctggctacacagcccgggtgcacgagatgttccaggta1661thrgluleualaglytyrthralaargvalhisglumetpheglnval410415420425tttgaagatgttcagcgctgtcacttcaagaggcccagggagctagag1709phegluaspvalglnargcyshisphelysargproarggluleuglu430435440gacgctcaggcggggtctgggaccataggccggtctggtgtccgtgtg1757aspalaglnalaglyserglythrileglyargserglyvalargval445450455gagggccccctgaagatccgaggccaggtggtggatgtggaacagggg1805gluglyproleulysileargglyglnvalvalaspvalgluglngly460465470atcatctgcgagaacatccccatcgtcacgccctcaggagaggtggtg1853ileilecysgluasnileproilevalthrproserglygluvalval475480485gtggccagcctcaacatcagggtggaggaaggcatgcatctgctcatc1901valalaserleuasnileargvalglugluglymethisleuleuile490495500505acaggccccaatggctgcggcaagagctccctgttccggatcctgggt1949thrglyproasnglycysglylysserserleupheargileleugly510515520gggctctggcccacgtacggtggtgtgctctacaagcccccaccccag1997glyleutrpprothrtyrglyglyvalleutyrlysproproprogln525530535cgcatgttctacatcccgcagaggccctacatgtctgtgggctccctg2045argmetphetyrileproglnargprotyrmetservalglyserleu540545550cgtgaccaggtgatctacccggactcagtggaggacatgcaaaggaag2093argaspglnvaliletyrproaspservalgluaspmetglnarglys555560565ggctactcggagcaggacctggaagccatcctggacgtcgtgcacctg2141glytyrsergluglnaspleuglualaileleuaspvalvalhisleu570575580585caccacatcctgcagcgggagggaggttgggaggctatgtgtgactgg2189hishisileleuglnarggluglyglytrpglualametcysasptrp590595600aaggacgtcctgtcgggtggcgagaagcagagaatcggcatggcccgc2237lysaspvalleuserglyglyglulysglnargileglymetalaarg605610615atgttctaccacaggcccaagtacgccctcctggatgaatgcaccagc2285metphetyrhisargprolystyralaleuleuaspglucysthrser620625630gccgtgagcatcgacgtggaaggcaagatcttccaggcggccaaggac2333alavalserileaspvalgluglylysilepheglnalaalalysasp635640645gcgggcattgccctgctctccatcacccaccggccctccctgtggaaa2381alaglyilealaleuleuserilethrhisargproserleutrplys650655660665taccacacacacttgctacagttcgatggggagggcggctggaagttc2429tyrhisthrhisleuleugl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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 745 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metprovalleuserargproargprotrpargglyasnthrleulys151015argthralavalleuleualaleualaalatyrglyalahislysval202530tyrproleuvalargglncysleualaproalaargglyleuglnala354045proalaglygluprothrglnglualaserglyvalalaalaalalys505560alaglymetasnargvalpheleuglnargleuleutrpleuleuarg65707580leuleupheproargvalleucysarggluthrglyleuleualaleu859095hisseralaalaleuvalserargthrpheleuservaltyrvalala100105110argleuaspglyargleualaargcysilealaarglysaspproarg115120125alapheglytrpglnleuleuglntrpleuleuilealaleuproala130135140thrphevalasnseralaileargtyrleugluglyglnleualaleu145150155160serpheargserargleuvalalahisalatyrargleutyrpheser165170175glnglnthrtyrtyrargvalserasnmetaspglyargleuargasn180185190proaspglnserleuthrgluaspvalvalalaphealaalaserval195200205alahisleutyrserasnleuthrlysproleuleuaspvalalaval210215220thrsertyrthrleuleuargalaalaargserargglyalaglythr225230235240alatrpproseralailealaglyleuvalvalpheleuthralaasn245250255valleuargalapheserprolyspheglygluleuvalalagluglu260265270alaargarglysglygluleuargtyrmethisserargvalvalala275280285asnserglugluilealaphetyrglyglyhisgluvalgluleuala290295300leuleuglnargsertyrglnaspleualaserglnileasnleuile305310315320leuleugluargleutrptyrvalmetleugluglnpheleumetlys325330335tyrvaltrpseralaserglyleuleumetvalalavalproileile340345350thralathrglytyrsergluseraspalaglualavallyslysala355360365alaleuglulyslysgluglugluleuvalsergluargthrgluala370375380phethrilealaargasnleuleuthralaalaalaaspalaileglu385390395400argilemetsersertyrlysgluvalthrgluleualaglytyrthr405410415alaargvalhisglumetpheglnvalphegluaspvalglnargcys420425430hisphelysargproarggluleugluaspalaglnalaglysergly435440445thrileglyargserglyvalargvalgluglyproleulysilearg450455460glyglnvalvalaspvalgluglnglyileilecysgluasnilepro465470475480ilevalthrproserglygluvalvalvalalaserleuasnilearg485490495valglugluglymethisleuleuilethrglyproasnglycysgly500505510lysserserleupheargileleuglyglyleutrpprothrtyrgly515520525glyvalleutyrlysproproproglnargmetphetyrileprogln530535540argprotyrmetservalglyserleuargaspglnvaliletyrpro545550555560aspservalgluaspmetglnarglysglytyrsergluglnaspleu565570575glualaileleuaspvalvalhisleuhishisileleuglnargglu580585590glyglytrpglualametcysasptrplysaspvalleuserglygly595600605glulysglnargileglymetalaargmetphetyrhisargprolys610615620tyralaleuleuaspglucysthrseralavalserileaspvalglu625630635640glylysilepheglnalaalalysaspalaglyilealaleuleuser645650655ilethrhisargproserleutrplystyrhisthrhisleuleugln660665670pheaspglygluglyglytrplyspheglulysleuaspseralaala675680685argleuserleuthrgluglulysglnargleugluglnglnleuala690695700glyileprolysmetglnargargleuglngluleucysglnileleu705710715720glyglualavalalaproalahisvalproalaproserproglngly725730735proglyglyleuglnglyalaserthr740745 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 659 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 3 : metalaalapheserlystyrleuthralaargasnserserleuala151015glyalaalapheleuleuleucysleuleuhislysargargargala202530leuglyleuhisglylyslysserglylysproproleuglnasnasn354045glulysgluglylyslysgluargalavalvalasplysvalphephe505560serargleuileglnileleulysilemetvalproargthrphecys65707580lysgluthrglytyrleuvalleuilealavalmetleuvalserarg859095thrtyrcysaspvaltrpmetileglnasnglythrleuilegluser100105110glyileileglyargserarglysaspphelysargtyrleuleuasn115120125pheilealaalametproleuileserleuvalasnasnpheleulys130135140tyrglyleuasngluleulysleucyspheargvalargleuthrlys145150155160tyrleutyrgluglutyrleuglnalaphethrtyrtyrlyslysgly165170175asnleuaspasnargilealaasnproaspglnleuleuthrglnasp180185190valglulysphecysasnservalvalaspleutyrserasnleuser195200205lyspropheleuaspilevalleutyrilephelysleuthrserala210215220ileglyalaglnglyproalasermetmetalatyrleuvalvalser225230235240glyleupheleuthrargleuargargproileglylysmetthrile245250255thrgluglnlystyrgluglyglutyrargtyrvalasnserargleu260265270ilethrasnserglugluilealaphetyrasnglyasnlysargglu275280285lysglnthrvalhisservalphearglysleuvalgluhisleuhis290295300asnpheileleupheargphesermetglypheileaspserileile305310315320alalystyrleualathrvalvalglytyrleuvalvalserargpro325330335pheleuaspleuserhisproarghisleulysserthrhisserglu340345350leuleugluasptyrtyrglnserglyargmetleuleuargmetser355360365glnalaleuglyargilevalleualaglyargglumetthrargleu370375380alaglyphethralaargilethrgluleumetglnvalleulysasp385390395400leuasnhisglylystyrgluargthrmetvalserglnglnglulys405410415glyilegluglyvalglnvalileproleuileproglyalaglyglu420425430ileileilealaaspasnileilelyspheasphisvalproleuala435440445thrproasnglyaspvalleuileargaspleuasnphegluvalarg450455460serglyalaasnvalleuilecysglyproasnglycysglylysser465470475480serleupheargvalleuglygluleutrpproleupheglyglyarg485490495leuthrlysprogluargarglysleuphetyrvalproglnargpro500505510tyrmetthrleuglythrleuargaspglnvaliletyrproaspgly515520525arggluaspglnlysarglysglyileseraspleuvalglnlysglu530535540tyrleuaspasnvalglnleuglyhisileleugluarggluglygly545550555560trpaspservalglnasptrpmetaspvalleuserglyglyglulys565570575glnargmetalametalaargleuphetyrhislysproglnpheala580585590ileleuaspglucysthrseralavalservalaspvalgluglytyr595600605iletyrserhiscysarglysvalglyilethrleuphethrvalser610615620hisarglysserleutrplyshishisglutyrtyrleuhismetasp625630635640glyargglyasntyrgluphelysglnilethrgluaspthrvalglu645650655pheglyser ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 4 : aggtggggcaggttggggtgccgggcacggagggaagcgtgtggcagggaggcccggggg60caggcagccgtgagcggtggggacagtctggggcgggccggggctgatgccaaaggtgtg120ggcaggccatgggagagccgggctggggtggg152 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 5 : cacccaatcgtaacctctggctctcggccttctgatggccaccatggcacagcgtgtgtg60agtggcactgggagaccctgaccatcgcccccacgggagctgcccctgtgcatggccagg120aagcctctctgtgtctgtcaccccccgcaggt152 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 6 : aggtgagacccagggctccaagaggatccaggccaggggcctgtcccccataccgctggg60tgctgagctcacgagggcccaactcagccagcccgccgcccacttctgctgccggggcca120ccgaggccctgctgccagccttgatgctttca152 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 7 : gcacatagagagaaagagagagagagctggttgccccggcaccatttgcagaagagcctc60gcctttctctccagcggctcatttttgactttccgctgtctctgccctgcccctccccgc120cccgccacccacccctctggggctttgcagat152 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 97 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 8 : aggaggtacccctggcccagccccacccttgccatccttgccatgcttctctccctgcaa60ctggcaggggctgagccagggtcaccctccctcaggt97 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 9 : aggtaaggctgtcccctccctatgagtgaccccgcccctgctgctgctgcaggtgctgac60ctgctgccccagctcctcctattcccgctccctcactcagggacctccatgtgcttctgg120cccatcccagtccacccaggacgggagggctg152 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 10 : ctggaccacaggctgctggtcaggaaccagctggcatgctgccagggatgggaatgaggg60cgtgcagccaggggcacgcagactccccagaatgcagaggggtcgccaccactccctctc120caccccagccccgctgtgctgtctctgcaggc152 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 11 : gggtaggtccagcggggagggcgccagccacgcacatatgcaagcctcagcccttggctt60cccgcctgtctgtgctggcaacagccattgtccctagatgtacgtggcaggtgggccaag120gtcaaggtgagagaccaacgtgtctctgactg152 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 12 : tcccccaggccctgctgtcccttatcaagagatcaagaatggcctgcgtgctggcctcgg60gcattgggagcctctcaaggctggtcaggaggccatagggtacgggaaggggcctgcgct120ctctggcgtcagcggctgttgcccctgcaggt152 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 340 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 13 : aggtaaggaagcccgtgcgcctctcctccacctcttcctgcctgtgcgctcacacatggc60ttcctgcagaggcccaggaagtggtgaagagtcagcacctcaggagaggacactgaggca120ctgtccccagagccagagacgggctgtggttcctgctccctccaaacccgcccgatccac180tgccctgttttggatctgtgtggggtgtgtgcacgggcggcgatgtgagcgtgtggatgc240gtgtgagcgtggcatgtggacactgcctgggaggcgcagagtatcttgggggaggcagag300ccggcccttccctccgtggacacccagctttcccacaggc340 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 14 : aggtaggaggcctggggctggcagccaccctttgtcccaccctggcctctcccttggcct60ccagggagtgaagattacctcaacatccagagtctaaagtgccaggtgccacggggcggg120gcagaggctgctaccagggaggaccaacacca152 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 152 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 15 : atgattaatgcctgtcagacagacaaggacgcagaggcacaggggccctgtcgtcacagc60tagctcattcccgcagctcccccagctccccggctggcccccgggtctgggtgctggtgg120aactgagccaagaccattgcccccgcctaggt152 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 153 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 16 : aggtgagcactccggaccggcaggctccctggggtcccctggaaggggaagtagcagctg60tggggaggcctgggctcagtggagcctgagccgggctggggtgttgggccctggagggtg120cacagactctcctctcggcccggacccccaggc153 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 146 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 17 : tggtaggtgccctgtctccctgcctggggtcggtgggagtgcctgcctgaggggaggagg60tggcctggcgggcccggcagcagcaggcggctgtcatcagcagcccccgtgccgtgcccc120tgaccctgtccctctcctggccagga146 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 1441 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 18 : gcggagcggacggacgcgcctggtgccccggggaggggcgccaccgggggaggaggagga60ggagaaggtggagaggaagagacgccccctctgcccgagacctctcaaggccctgacctc120aggggccagggcactgacaggacaggagagccaagttcctccacttgggctgcccgaaga180ggccgcgaccctggagggccctgagcccaccgcaccaggggccccagcaccaccccgggg240gcctaaagcgacagtctcaggggccatcgcaaggtttccagttgcctagacaacaggccc300agggtcagagcaacaatccttccagccacctgcctcaactgctgccccaggcaccagccc360cagtccctacgcggcagccagcccaggtgacatgccggtgctctccaggccccggccctg420gcgggggaacacgctgaagcgcacggccgtgctcctggccctcgcggcctatggagccca480caaagtctaccccttggtgcgccagtgcctggccccggccaggggtcttcaggcgcccgc540cggggagcccacgcaggaggcctccggggtcgcggcggccaaagctggcatgaaccgggt600attcctgcagcggctcctgtggctcctgcggctgctgttcccccgggtcctgtgccggga660gacggggctgctggccctgcactcggccgccttggtgagccgcaccttcctgtcggtgta720tgtggcccgcctggacggaaggctggcccgctgcatcgcccgcaaggacccgcgggcttt780tggctggcagctgctgcagtggctcctcatcgccctccctgctaccttcgtcaacagtgc840catccgttacctggagggccaactggccctgtcgttccgcagccgtctggtggcccacgc900ctaccgcctctacttctcccagcagacctactaccgggtcagcaacatggacgggcggct960tcgcaaccctgaccagtctctgacggaggacgtggtggcctttgcggcctctgtggccca1020cctctactccaacctgaccaagccactcctggacgtggctgtgacttcctacaccctgct1080tcgggcggcccgctcccgtggagccggcacagcctggccctcggccatcgccggcctcgt1140ggtgttcctcacggccaacgtgctgcgggccttctcgcccaagttcggggagctggtggc1200agaggaggcgcggcggaagggggagctgcgctacatgcactcgcgtgtggtggccaactc1260ggaggagatcgccttctatgggggccatgaggtggggcaggttggggtgccgggcacgga1320gggaagcgtgtggcagggaggcccgggggcaggcagccgtgagcggtggggacagtctgg1380ggcgggccggggctgatgccaaaggtgtgggcaggccatgggagagccgggctggggtgg1440g1441 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 481 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 19 : cacccaatcgtaacctctggctctcggccttctgatggccaccatggcacagcgtgtgtg60agtggcactgggagaccctgaccatcgcccccacgggagctgcccctgtgcatggccagg120aagcctctctgtgtctgtcaccccccgcaggtggagctggccctgctacagcgctcctac180caggacctggcctcgcagatcaacctcatccttctggaacgcctgtggtatgttatgctg240gagcagttcctcatgaagtatgtgtggagcgcctcgggcctgctcatggtggctgtcccc300atcatcactgccactggctactcagagtcaggtgagacccagggctccaagaggatccag360gccaggggcctgtcccccataccgctgggtgctgagctcacgagggcccaactcagccag420cccgccgcccacttctgctgccggggccaccgaggccctgctgccagccttgatgctttc480a481 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 702 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 20 : gcacatagagagaaagagagagagagctggttgccccggcaccatttgcagaagagcctc60gcctttctctccagcggctcatttttgactttccgctgtctctgccctgcccctccccgc120cccgccacccacccctctggggctttgcagatgcagaggccgtgaagaaggcagccttgg180aaaagaaggaggaggagctggtgagcgagcgcacagaagccttcactattgcccgcaacc240tcctgacagcggctgcagatgccattgagcggatcatgtcgtcgtacaaggaggtacccc300tggcccagccccacccttgccatccttgccatgcttctctccctgcaactggcaggggct360gagccagggtcaccctccctcaggtgacggagctggctggctacacagcccgggtgcacg420agatgttccaggtatttgaagatgttcagcgctgtcacttcaagaggcccagggagctag480aggacgctcaggcggggtctgggaccataggccggtctggtgtccgtgtggagggccccc540tgaagatccgaggtaaggctgtcccctccctatgagtgaccccgcccctgctgctgctgc600aggtgctgacctgctgccccagctcctcctattcccgctccctcactcagggacctccat660gtgcttctggcccatcccagtccacccaggacgggagggctg702 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 395 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 21 : ctggaccacaggctgctggtcaggaaccagctggcatgctgccagggatgggaatgaggg60cgtgcagccaggggcacgcagactccccagaatgcagaggggtcgccaccactccctctc120caccccagccccgctgtgctgtctctgcaggccaggtggtggatgtggaacaggggatca180tctgcgagaacatccccatcgtcacgccctcaggagaggtggtggtggccagcctcaaca240tcagggtaggtccagcggggagggcgccagccacgcacatatgcaagcctcagcccttgg300cttcccgcctgtctgtgctggcaacagccattgtccctagatgtacgtggcaggtgggcc360aaggtcaaggtgagagaccaacgtgtctctgactg395 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 928 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 22 : tcccccaggccctgctgtcccttatcaagagatcaagaatggcctgcgtgctggcctcgg60gcattgggagcctctcaaggctggtcaggaggccatagggtacgggaaggggcctgcgct120ctctggcgtcagcggctgttgcccctgcaggtggaggaaggcatgcatctgctcatcaca180ggccccaatggctgcggcaagagctccctgttccggatcctgggtgggctctggcccacg240tacggtggtgtgctctacaagcccccaccccagcgcatgttctacatcccgcagaggtaa300ggaagcccgtgcgcctctcctccacctcttcctgcctgtgcgctcacacatggcttcctg360cagaggcccaggaagtggtgaagagtcagcacctcaggagaggacactgaggcactgtcc420ccagagccagagacgggctgtggttcctgctccctccaaacccgcccgatccactgccct480gttttggatctgtgtggggtgtgtgcacgggcggcgatgtgagcgtgtggatgcgtgtga540gcgtggcatgtggacactgcctgggaggcgcagagtatcttgggggaggcagagccggcc600cttccctccgtggacacccagctttcccacaggccctacatgtctgtgggctccctgcgt660gaccaggtgatctacccggactcagtggaggacatgcaaaggaagggctactcggagcag720gacctggaagccatcctggacgtcgtgcacctgcaccacatcctgcagcgggagggaggt780aggaggcctggggctggcagccaccctttgtcccaccctggcctctcccttggcctccag840ggagtgaagattacctcaacatccagagtctaaagtgccaggtgccacggggcggggcag900aggctgctaccagggaggaccaacacca928 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 1025 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 23 : atgattaatgcctgtcagacagacaaggacgcagaggcacaggggccctgtcgtcacagc60tagctcattcccgcagctcccccagctccccggctggcccccgggtctgggtgctggtgg120aactgagccaagaccattgcccccgcctaggttgggaggctatgtgtgactggaaggacg180tcctgtcgggtggcgagaagcagagaatcggcatggcccgcatgttctaccacaggtgag240cactccggaccggcaggctccctggggtcccctggaaggggaagtagcagctgtggggag300gcctgggctcagtggagcctgagccgggctggggtgttgggccctggagggtgcacagac360tctcctctcggcccggacccccaggcccaagtacgccctcctggatgaatgcaccagcgc420cgtgagcatcgacgtggaaggcaagatcttccaggcggccaaggacgcgggcattgccct480gctctccatcacccaccggccctccctgtggtaggtgccctgtctccctgcctggggtcg540gtgggagtgcctgcctgaggggaggaggtggcctggcgggcccggcagcagcaggcggct600gtcatcagcagcccccgtgccgtgcccctgaccctgtccctctcctggccaggaaatacc660acacacacttgctacagttcgatggggagggcggctggaagttcgagaagctggactcag720ctgcccgcctgagcctgacggaggagaagcagcggctggagcagcagctggcgggcattc780ccaagatgcagcggcgcctccaggagctctgccagatcctgggcgaggccgtggccccag840cgcatgtgccggcacctagcccgcaaggccctggtggcctccagggtgcctccacctgac900acaaccgtccccggcccctgccccgcccccaagctcggatcacatgaaggagacagcagc960acccacccatgcacgcaccccgcccctgcatgcctggcccctcctcctagaaaacccttc1020ccgcc1025__________________________________________________________________________