Patent Application: US-70184696-A

Abstract:
this disclosure relates to an isolated and cloned dna from a granulovirus virus which comprises an amino acid sequence of the vital gene encoding a polypeptide isolated from occlusion bodies of certain baculoviruses and which polypeptide possesses the biological activity of enhancing baculovirus infectivity . such proteins termed herein as &# 34 ; enhancins &# 34 ; are found within the viral occlusion body , have a disruptive effect on the insect peritrophic membrane proteins , and / or interact with the midgut epithelium in such a manner as to permit the increased adsorption , penetration and uptake of virus particles by midgut cells with a concomitant increase in host mortality . disclosed herein is a recombinant dna sequence which codes for the enhancin protein of the helicoverpa armigera granulovirus virus . the dna sequence is shown in seq . id . no . : 1 and the open reading frame is shown in seq . id . no . : 1 : base pairs 271 - 2976 . the amino acid sequence of the enhancin protein is shown in seq . id . no . : 2 .

Description:
one of the discoveries of the present invention is the cloning and sequencing of the enhancin gene found in the helicoverpa ( heliothis ) armigera granulovirus ( heargv ). while , the enhancin gene found in the psungv - h virus is virtually identical to the previously characterized trichoplusia ni gv ( tngv ) enhancin gene , a comparison of the predicted amino - acid ( aa ) sequences of tngv enhancin ( 901 aa ) with the heargv enhancin ( 902 aa ) revealed herein demonstrated an overall identity of only 80 %, with greater conservation ( 88 %), and hence higher homology , from amino - acids 1 - 550 . the addition of the newly discovered heargv enhancin to an acmnpv inoculom increased the infectivity of baculoviruses in insect larvae . this discovery will aid in the effort to control certain pests without the use of pesticides . moreover , the host range of the helicoverpa ( heliothis ) armigera granulovirus is significantly broader than that of other known granulovirus viruses , such as tngv or pugv , which suggests that the heargv enhancin disclosed herein may have broader biological activity , increasing its potential effectiveness in the control of insects ( john j . harem , 1982 , incorporated herein by reference ). a pathogen , here a insect baculovirus , that is an effective microbial control against several species of economically damaging insects , as is heargv due to its broad specificity , is much more useful against a complex of insects attacking a crop and therefore more economically viable as a commercially useful insect pathogen than a similar enhancin with a narrower insect target range . proteins from eight different granuloviruses that infected 4 different lepidopteran families were analyzed by sds - page and western blotting in an attempt to identify novel enhancin proteins . the granulovirus ( gv ) species used in this study were : cpgv , estigmene acrea ( esacgv ), heliocoverpa armigera ( heargv ), plingv , pieris rapae ( piragv ), psungv - h , scotogramma trifiolii ( sctrgv ) and tngv . viral stocks were maintained in the lab as granule suspensions and / or infectious hemolymph isolated from previously infected larvae . larval infections , purification of granules and enhancin was as previously described ( derksen & amp ; granados , 1988 ; greenspan & amp ; gallo et at ., 1991 , both incorporated herein by reference ). for the sds - page and western blot analysis granules were dissolved in 100 mm nahco 3 ( ph 10 . 5 ) and incubated at room temperature for 15 minutes . the protein concentration was determined using the bradford assay kit ( promega corp ., madison , wi ). approximately 10 μg of each gv solution was analyzed on sds - page . gels were silver stained . a duplicate gel was transferred onto pvdf membrane ( nen research products , dupont , boston , ma ) using a protocol provided by the manufacturer ( biorad , melville , ny ). western blots were analyzed using a rabbit anti - vef - trpe polyclonal antibody at a dilution of 1 : 5000 . cross - reactive bands were visualized using goat anti - rabbit alkaline phosphatase conjugated secondary antibody , this was done at a dilution of 1 : 3000 ( sigma , st . louis , mo ). five different gv species , four infecting the noctuidae family and one infecting the pieridae family , were found to contain proteins that did cross - react with the anti - enhancin polyclonal antibodies already mentioned . however , plingv , which infects indian meal moth larvae ( noctuidae ), cpgv , that infects codling moth larvae ( torticidae ) and esacgv , that infects saltmarsh caterpillar larvae ( arctiidae ), were not seen to have crossreacting proteins . two granulovirus species , c . pomonella gv , and e . acrea gv , were previously identified as having an enhancin , and were used as positive controls in the research herein disclosed . we also used standard neonate bioassays performed with both eagv and cpgv , in attempt to demonstrate enhancin activity therein , however , neither displayed any ability to enhance the infectivity of baculovirus . the enhancins can be subdivided into of three different groups based on their respective molecular weights , and migration patterns : 104 kd for the tngv , psungv - h and piragv enhancins , 108 - 110 kd for heargv enhancin and 120 kd for sctrgv enhancin . in standard t . ni neonate bioassays heargv , psungv - h and sctrgv demonstrated an ability to &# 34 ; enhance &# 34 ; the level acmnpv infectivity , confirming the presence of an enhancin . psungv - h , was the first granulovirus for which the presence of an enhancin in its occlusion body was described and documented . vital genomic dna was isolated from granules as described by smith and summers ( 1982 ), incorporated herein by reference . all of the restriction endonucleases and modifying enzymes were purchased from promega corp . ( madison , wi ). restriction endonucleases were routinely used in dna digests using a universal enzyme restriction endonuclease buffer ( 10x = 0 . 33 m tris / acetate ph 7 . 85 , 0 . 65 m potassium acetate , 0 . 1 m magnesium acetate , 0 . 04 m spermidine tri - chloride , 5 mm dithiothreitol ). dna gel electrophoresis , southern blotting and hybridization were performed as described ( hashimoto et al ., 1991 ). viral genomic dna was cloned into either pljc18 / 19 ( yanischperron et at ., 1985 ) or bluescript sk ( stratagene , la jolla , ca ). the complete heargv enhancin gene was identified through hybridization of a 1 . 75 kb kpni fragment of the heargv gene , that crosshybridized to a tngv internal enhancin fragment on a blot , where the heargv genomic dna had been digested with bamhi , ecori , hindlii and kpni . a cross - hybridizing bamhi fragment with an estimated size of 5 . 2 kb was cloned into the puc18 expression vector and a dna restriction map was thereafter generated . the kpni fragment already mentioned , as well as the 1 . 45 kb sstii - bamhi fragment , and deletion fragments derived thereof , were sequenced on both strands . heargv genomic clones were sequenced using cesium chloride purified dna and a commercially available sequencing kit ( united states biochemical corp ., cleveland , oh ). deletion clones were generated by bal31 digestion as described , the erase - a - base exolii / s1 digestion kit ( promega corp ., madison , wi ) or an exolii / mung bean nuclease kit ( stratagene ). sequence products were analyzed on gels prepared with 6 % sequagel rapid sequencing solution ( national diagnostics , atlanta , ga ). sequence information from the generated genomic clones was developed through the use of the sequence analysis software package of the genetics computer group ( madison , wi , versions 7 . 2 and 7 . 3 ). the enhancin gene from heargv was cloned and sequenced as described above . every nucleotide on both strands of the enhancin gene sequence was sequenced a minimum of 2 times . the dna sequence and deduced amino acid sequence of the heargv gene is shown in seq . id . no : 1 and seq . id . no : 2 , respectively . sequence data analysis of the heargv dna revealed an open reading frame ( orf ) of 2706 nucleotides that encodes a protein containing 902 amino acids and a mass of 104 . 6 kd . similarly , psungv - h clones have an orf of 2703 bp that encodes a protein of 901 amino acids with a mass of 104 . 2 kd . ( please note that the dna and amino acid sequence for psungv - h and tngv enhancins are presented in the sequence listing of u . s . pat . no . 5 , 475 , 090 ). total rna was isolated from heargv infected t . ni larvae at dally intervals from 0 - 8 days post infection ( p . i . ), using guanidine isothiocyanate ( git ) as described ( sambrook et at ., 1989 inc . by ref .). four to eight larvae were collected for each timepoint , frozen in liquid nitrogen and then ground in a glass potter tube in the presence of 4 ml of git . the resulting larval suspension was layered onto a csci gradient and spun for 16 hours at 35 k . the rna pellet was then collected , precipitated , washed , dried and quantified . for primer extension analysis of the enhancin promoter , a primer ( hazr2 ) 5 &# 39 ; cac ggc ggc agc acg g 3 &# 39 ; complementary to nucleotides 43 - 28 downstream of the aug initiator codon of the enhancin gene was used . approximately 100 ng of the primer was labeled using 100 mci of g - atp ( dupont company , boston , ma ) and t4 polynucleotide kinase ( promega corp ., madison . wi ). five nanograms of the primer were incubated with 50 μg of total rna isolated from heargv infected t . ni larvae isolated at 1 , 4 , 5 , 6 , 7 and 8 days post infection . the primer extension reaction was according to ausubel et at ., ( 1989 inc . by ref .) with two modifications : the amv reverse transcriptase ( rvt ) ( promega , madison , wi ) was incubated at 50 ° c . and actinomycin d was added at a final concentration of 75 μg / ml to inhibit the dna - dependent dna polymerase activity of the rvt . reaction products were analyzed on a 6 % polyacrylamide ( paa )- gel and compared to a sequencing ladder of clone habam , that contains a 5 . 2 kb bamhi fragment from the heargv genome and has the complete enhancin coding sequence also sequenced with the same primer . for the analysis of the 5 &# 39 ; end of the heargv enhancin message , a 800 bp ncoi fragment from clone habam was subcloned in vector psl1180 ( pharmacia , piscataway , nj ) to yield phanco . a 470 bp muni - bamhi fragment ( 425 bp heargv sequences and 45 bp psl1180 multilinker ) was subcloned into bluescript ks + digested with ecori and bamhi to yield phamb . to generate a probe complementary to the 5 &# 39 ; end of the heargv enhanein gene , plasmid phamb was linearized at the hindiii multilinker site , which lies upstream of the muni / ecori fusion site . transcription with t7 - rna polymerase ( gibco brl , gaithersburg , md ) was according to the manufacturer &# 39 ; s protocol in the presence of 20 μci utp . after transcription , the template was digested for 15 minutes at 37 ° c . by adding 2 μl rnase free dnase ( 10 , 000 u / ml ; boehringer mannheim , indianapolis , in ). following digestion , 100 μl tse ( te plus 0 . 5 % sds ) was added , followed by extraction with pci ( phenol / chloroform / isoamyl alcohol , 25 : 24 : 1 ). the probe was further purified by elution from a nick g - 50 gravity flow column ( pharmacia , piscataway , nj ). two microliters of the resulting purified probe were analyzed on a 6 % polyacrylamide - gel . the presence of several premature stops further necessitated purification on a 6 % paa gel . the full length probe ( 530 nucleotides ) was isolated from this gel . for analysis of the 3 &# 39 ; end of the enhancin message , clone hasb , which has a 1 . 45 kbp sstii - bamhi fragment of the heargv enhancin gene cloned into bluescript sk -, was cut with mlui . an antisense t7 rna probe was synthesized and purified as described above . upon gel analysis the probe was found to be & gt ; 95 % full length ( 372 nucleotides ) and used without further purification . the heargv gene inserted into the plasmid vector was transfected into e . coli and deposited under the budapest treaty on aug . 22 , 1996 at the agricultural research service culture collection ( nrrl ), northern regional research center , agricultural research service , u . s . department of agriculture , 1815 north university street , peoria , ill . 61604 and assigned accession number : nrrl b - 21614 . the applicant agrees to be bound by the terms of the budapest treaty regarding availability . for rnase protection analysis , 20 μg of total rna were mixed with 105 cpm of antisense rna probe , precipitated and resuspended in 30 μl of hybridization buffer ( 80 % formamide , 40 mm pipes , 400 mm nacl , 1 mm edta ). after heating for 10 minutes at 80 ° c ., the mixture was left to hybridize overnight at 50 ° c . following hybridization , unprotected rna was digested by adding 350 μl rnase mix ( 10 mm tris ph 7 . 5 , 5 mm edta , 0 . 3 m nacl , 2 μl 10 mg / ml rnase a , 5 μl 100 , 000 u / ml rnase t1 ) and incubated at 30 ° c . for 60 minutes . the digestion was stopped by adding sds to a final concentration of 0 . 5 %. samples were further purified by digestion with proteinase k ( 3 μl of a 15 mg / ml premix ; boehringer mannheim ) for 30 minutes at 37 ° c . the samples were extracted twice with 400 μl pci and precipitated at room temperature ( rt ) for 30 minutes by addition of 1 ml of 96 % ethanol . samples were spun at room temperature , washed twice with 70 % ethanol at room temperature , dried briefly and resuspended in 20 μl loading buffer ( 50 % formamide , bfb and xcff 0 . 05 %). reaction products were analyzed on 6 % polyacrylamide sequencing ( 3 &# 39 ; analysis ) or slab gels ( 5 &# 39 ; analysis ). product sizes were estimated by comparison to a sequencing ladder of clone habam sequenced with primer hazr2 and comparison to a 123 bp ladder ( gibco brl ). gels were exposed to xar 5 film in cassettes with intensifying screens ( kodak , rochester , ny ). the rnase protection analysis of the 3 &# 39 ; end of the heargv enhancin offers some evidence both for transcription termination and transcriptional read through into the second orf . no canonical polyadenylation signal aauaaa , was present upstream of the transcription termination signal . an alternative poly ( a ) sequence closely resembling the poly ( a ) sequence , aacaaa , was present between nucleotides 3003 - 3008 . it has been reported that the presence of a similar sequence downstream of the β - thalassaemia gene or other alternative poly ( a ) like sequences results in elongated and unstable transcripts and changes to the steady state levels of mrna . this may explain why , apart from the two major protected rna species during rnase protection , several minor protected species appeared to be protected . alternatively , it is possible that the heargv enhancin gene transcript does not have a poly ( a ) tail . this would not be unique for baculoviruses . recently , a convincing case has been made for the absence of a poly ( a ) tail on spodoptera exigua npv polyhedrin mrna . the 3 &# 39 ; rnase protection assay also indicated that the enhancin orf and the downstream orf maybe on one message . the occurrence of bi - cistronic messengers is not uncommon for baculoviruses . analysis of the hindlii - m region of orgyia pseudotsugata npv for instance , has shown that hi ,- and multi - cistronic messages originated from baculovirus late promoter motifs that had different 5 &# 39 ; ends but the same 3 &# 39 ; end . transcriptional analysis of the heargv gene reveals several more interesting features of enhancin genes . of the three baculovirus late promoter motifs conforming to the consensus ntaag sequence , present in the region upstream of the enhancin orf the one that is predominantly used , ttaag , is positioned only 3 nucleotides from the translational aug start codon . transcriptional analysis of the tngv enhancin gene has also shown that the ataag motif present at - 8 to - 4 relative to the aug initiator codon serves as the only transcriptional initiation point . from our work it has become clear that the aug closest to the promoter is the translation initiation codon . that this codon is the translation initiator codon is based on several observations seen infra . first , the context of the initiator codon - 3 aucaugc + 4 is similar to the kozak consensus sequence for translation initiation , - 3 a / gyyaugg + 4 . the pyrimidine c present at position + 4 in the heargv gene , and in all enhancin genes sequenced so far , is the only notable exception to the kozak consensus . it has been found through mutation studies that replacement of the consensus g in position + 4 acts to downregulate eukaryotic translation . second , the mass of the protein observed in protein gels corresponds well with the first aug in the orf acting as the translational initiator codon . third , the next possible aug lies 240 nucleotides downstream in the coding region and does not confer to kozak rules at all . the observation that a baculovirus late promoter in the heargv gene is present so proximal to the start codon that upon transcription it gives rise to a 5 &# 39 ; leader with a maximum length of 7 nucleotides , is unique to the enhancin genes , and has only been previously reported for the tngv enhancin gene . leaders of eukaxyotic messages are seldom shorter than 7 - 10 nucleotides and usually average between 25 and 50 nucleotides . deletion studies of the 5 &# 39 ; non - coding region on the translational efficiency of phosphoglycerate kinase mrna in yeast , for instance , have shown that even if the leader length is decreased to 7 nucleotides , translation at 50 % of the optimal rate still occurs . the observation that a shorter leader impairs the fidelity of initiation by eukaryotic ribosomes , has been confirmed using an in vitro transcription and translation system ( kozak , 1991 , inc . by ref .). this negative effect on fidelity of initiation , however , can be almost completely eliminated by the presence , or through the introduction of , secondary structure with a dg of - 19 kcal / mol in the mrna at an optimal distance of 14 nucleotides from the aug initiator codon ( see , kozak , 1990 , 1991 ). transcription of the heargv enhancin gene also results in a short 5 &# 39 ; leader sequence with considerable secondary structure ( dg - 16 . 7 kcal / mol : gcg mfold analysis ) within the first 100 nucleotides of the mrna . the total amount of enhancin present in granules has been estimated to be 5 % of total protein . this suggests that enhanein gene promoters are rather strong . the combination of promoter proximity , suboptimal aug context and secondary structure downstream of the aug initiator codon , may very well result in a complex mechanism of transcription regulation , mrna stability , and translation efficiency unique to baculoviruses after sequencing the heargv enhancin gene , as laid out above , it was determined that it shared significant sequence homology with both the tngv and psungv - h enhancin genes ( see table 1 below ). analysis of this similarity in sequence between the psungv - h and heargv enhancin genes at the dna and deduced amino acid sequence level , and thereafter comparison with the tngv enhancin gene sequence revealed several interesting characteristics of the genes , and their respective homology . the tngv and psungv - h enhancin genes are virtually identical from 325 nucleotides upstream of the enhancin orf and throughout the partial orf identified downstream of the enhancin gene . since in hindiii digests of tngv and psungv - h genomic dna , 19 of the 26 visible fragments comigrate the observed conservation of homology suggests that both granuloviruses are strongly related and may have only recently evolved divergently . table 1__________________________________________________________________________comparison of the tngv , psungv - h and heargvdna and amino acid sequences__________________________________________________________________________nucleotide sequence identity upstream upstream upstream overall intergenic downstreamvirus compare 450 - 325 n 334 - 65 n 65 - 1 n atg - stop region orf__________________________________________________________________________ha × tn nd nd 40 % 77 % 46 % 73 % ha × pu nd 38 % 40 % 77 % 46 % 69 % pu × tn 35 % 94 % 100 % 99 % 100 % 98 % __________________________________________________________________________amino acid sequence identity overall amino acid amino acid downstream downstreamvirus compare 1 - end 1 - 551 551 - end orf orf 20 - end id sim id sim id sim id sim id sim__________________________________________________________________________ha × tn 80 % 90 % 88 % 94 % 68 % 83 % 82 % 89 % 93 % 98 % ha × pu 81 % 90 % 89 % 95 % 69 % 84 % 78 % 86 % 95 % 100 % pu × tn 98 % 99 % 99 % 99 % 97 % 99 % 94 % 97 % 100 % 100 % __________________________________________________________________________ nd : not determined id : identity sim : similarity n : nucleotide a comparison of the psungv - h and tngv enhancin genes ( fig1 & amp ; table 1 ), showed that they are virtually identical at both the dna and the amino acid sequence level over almost their entire length . the amino acid sequences differ in only fifteen amino acids . seven changes are caused by a 21 nucleotide reciprocal frameshift ( reflected by the dissimilarity in amino acids 652 - 658 , fig4 ). the remaining eight amino acid changes are caused by point mutations in the psungv - h enhancin gene . the dna homology from - 450 through - 325 nucleotides upstream of the translational initiator codon is 35 %. this increases to 94 % from - 325 to - 1 and 98 % for the remaining dna . there is a 98 % identity for the peptide sequences . when the heargv and psungv - h enhancin genes are compared , virtual identity is not present ( fig1 & amp ; table 1 ). for 270 nucleotides upstream of the translational start codon the homology is 40 %. this increases to an overall homology of 77 % within the orf , decreases to 46 % for the intergenic region and increases again to 69 % for the putative downstream orf . comparison of the amino acid sequences of the two enhancins revealed that the overall identity and similarity are 81 % and 90 % respectively . amino acids 1 - 550 show an identity and similarity of 89 % and 95 %. this similarity decreases to 69 % and 84 %, when amino acids 551 - 902 , respectively , are compared . while amino acid sequences 1 - 550 score significantly above the similarity average , the amino acids 551 - 901 scored significantly below it , when the gcg plotsim software program is used to generate a similarity comparison . the above analysis demonstrates that enhancins have two distinct domains . this may be a reflection of the fact that two activities have been found to be associated with enhancins , namely the breakdown of peritrophic membranes through a proteolytic activity and the enhancement of viral binding to receptors present in the midgut microvilli . the heargv gene has a consensus baculovirus late promoter motif shared with the psungv - h gene ( psungv - h : ataag ; heargv : ttaag ,) at positions - 8 through - 4 relative to the translational start codon . two other late motifs , gtaag and ataag , were present in the heargv sequence within 270 basepairs upstream of the atg . the psungv - h sequence has no other late promoter motifs within 400 nucleotides upstream of the aug . a second late promoter motif , ataag was present at 36 - 32 nucleotides upstream of the enhancin translational stop codon ( positions 2991 - 2993 ) with the start codon of a putative orf ( psungv - h : 3057 - 3059 ; heargv : 3051 - 3053 ) downstream of it . no canonical polyadenylation consensus sequences were identifiable in the short intergenic region between the translational stop codon of the enhancin gene and the translational start codon of the downstream orf . it was noted however , that the heargv dna sequence here is relatively at rich , and the psungv - h enhancin gene has two sequential stop condons , taataa . either of these dna sequences may meet the requirements for polyadenylation sites . the heargv enhancin gene has several baculovirus late promoters upstream of the enhancin orf and encodes a significantly different protein from previously described enhancins , one which differs in both mass and deduced amino acid sequence from the enhancin of tngv and psungv - h . therefore it is a separate and novel gene product from previously described genes . while the heargv enhancin has 902 amino acids and a mass of 104 . 6 kd , it migrates at a higher than expected mass of 108 - 110 kd . we hypothesize that this variant migration behavior may be caused by the presence of a sequential stretch of proline residues at the c - terminal end of this sequence ( positions 880 - 883 ), that is lacking from the psungv - h sequence . comparison of the partial amino acid sequence of the downstream orf ( heargv : aug = 3051 , psungv - h : aug = 3057 ) shows an identity and similarity of 82 and 89 %, respectively ( fig5 ; table 1 ). analysis of the amino acid sequences with a gcg program that recognizes signal sequences with a probability of 95 % and an accuracy of 75 % at a score level of 3 . 5 , revealed that the first twenty amino acids of both the heargv and the psungv - h downstream orf , most likely encode a similar signal sequence ( the signal cleave scores are 8 . 8 and 7 . 3 respectively ). the psungv - h downstream orf has been shown to be open for at least 170 amino acids . the organization , transcription and translation of these enhancin genes hold several special features that can be modulated to serve as useful alternatives to the application of chemical pesticides for insect management . as table 2 , below , demonstrates that the use of enhancin proteins , specifically the heargv enhancin protein , dramatically affects the mortality of host larvae ( see table 2 below ). also as seen in table 2 , the amounts of the enhancins needed to substantially increase insect host mortality are very small . table 2______________________________________the use of various enhancin proteins to increase the infectivity ofbaculoviruses , and thereby the mortality in treated insect larvaeenhancin percent mortalityconcentration tn ha st pu______________________________________trial 1 0 . 1 ng 100 % 100 % 100 % 100 % 0 . 01 ng 97 % 90 % 90 % 97 % trial 2 0 . 1 ng 90 % 100 % 97 % 100 % 0 . 01 ng 97 % 90 % 90 % 100 % ______________________________________ percent mortality in t . ni neonate larvae following ingestion of one acmnpv ob per larva at two different concentrations of enhancin derived from four different granulovirus viruses . acmnpv control at 1 ob / larva wa 50 % and 47 % for 2 replications ; tn = trichoplusia ni gv ; ha = heliothis armigera gv ; st = scotogramma trifolii gv ; pu = pseudaletia unipuncta gv . accordingly , it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention . reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims , which themselves recite those features regarded as essential to the invention . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 3186 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : circular ( ii ) molecule type : dna ( genomic )( iii ) hypothetical : no ( iv ) anti - sense : no ( vi ) original source :( a ) organism : helicoverpa armigera granulosis virus ( ix ) feature :( a ) name / key : cds ( b ) location : 271 .. 2976 ( xi ) sequence description : seq id no : 1 : ctatatgtggtaagattgattgaatgttcgtttgctctcgcgttgtttaaaaaacgcaac60aaacaccggtattagcggagcggtctcctgtatccggcagcggactgtcgcgttagcgtg120caccactacgccggccggaacgcgaccgtgtttgagcgccaaccaattgtcctgtggcct180caaataaggcggcactgctaaagatggtaccggtagaataatggaagttttgttctttca240cacaacattacgctgtcttaaattaagattatgtcgtacaacgtaattgtgcct294metsertyrasnvalilevalpro15actaccgtgctgccgccgtggctgaggatcggtcaaaattggatattc342thrthrvalleuproprotrpleuargileglyglnasntrpilephe101520gctagacacagacgcaccgaagtcggtgtggtgttacctgcaaacaca390alaarghisargargthrgluvalglyvalvalleuproalaasnthr25303540aagtttcgggttcgagccgatttcgctaaatggggcatcacgaggccc438lyspheargvalargalaaspphealalystrpglyilethrargpro455055gtgatcgtgcgcctcttgaacaacaaccgtaacaccgagcgcgagata486valilevalargleuleuasnasnasnargasnthrgluarggluile606570aatttaaccaacgaccaatggatagagatggagcacgagcacgagtgt534asnleuthrasnaspglntrpileglumetgluhisgluhisglucys758085gtgccgttcgtcgactggccggtgggtgaaaaaaacaccatggccgag582valprophevalasptrpprovalglyglulysasnthrmetalaglu9095100gtacactttgaaatcgacggaccacacataccgcttcccgtgtacgtg630valhisphegluileaspglyprohisileproleuprovaltyrval105110115120ttcaacacgagacccgtggaaaactttaagagcgagtaccgccagagt678pheasnthrargprovalgluasnphelysserglutyrargglnser125130135tcgtcgggctactgcttcctgtatttggacctggtgtgtattttggtg726serserglytyrcyspheleutyrleuaspleuvalcysileleuval140145150ccgccggctagtaaaaacgtgttactagacacggacctgtttgagctc774proproalaserlysasnvalleuleuaspthraspleuphegluleu155160165catcaattttataacgaaattattaattactatgacgatttgtgcggt822hisglnphetyrasngluileileasntyrtyraspaspleucysgly170175180ttggtcgaggacccgtacgcagacactgtggattcaaacctacccaac870leuvalgluaspprotyralaaspthrvalaspserasnleuproasn185190195200aaggcggcattcgtgaaagccgacggtggtggtcccggcggtgcttat918lysalaalaphevallysalaaspglyglyglyproglyglyalatyr205210215tacggggcattctggacggctcccgccagcacaaatctaggcgaatat966tyrglyalaphetrpthralaproalaserthrasnleuglyglutyr220225230ctccgggtgtcgcccaccaattggatggttattcacgagctgggtcac1014leuargvalserprothrasntrpmetvalilehisgluleuglyhis235240245gcgtacgatttcgtgtttactgtgaacactcgccttatagaaatctgg1062alatyraspphevalphethrvalasnthrargleuilegluiletrp250255260aacaactcgttctgcgatcggatacaatacacgtggatgaacaaaacc1110asnasnserphecysaspargileglntyrthrtrpmetasnlysthr265270275280aagcgacagcaactggctcgcatttacgagaaccaacgaccccagaag1158lysargglnglnleualaargiletyrgluasnglnargproglnlys285290295gaggcggctattcaagcgctaatcgacaacaatgtaccgtttgataat1206glualaalaileglnalaleuileaspasnasnvalpropheaspasn300305310tgggatttttttgagaaactcagcatttttgcatggctgtacaatccg1254trpaspphepheglulysleuserilephealatrpleutyrasnpro315320325caaaggggactggacactttgcgtaatatcaatcattcgtacaggttg1302glnargglyleuaspthrleuargasnileasnhissertyrargleu330335340cacgctgcccgcaatccagttacgccatacccgcaaatttgggcatgg1350hisalaalaargasnprovalthrprotyrproglniletrpalatrp345350355360ttgatgagttgtggttacgacaacttttggttgtactttaatcgaata1398leumetsercysglytyraspasnphetrpleutyrpheasnargile365370375ggtttgtaccctgccgatttttacattaacgaacacaataaagtcgtg1446glyleutyrproalaaspphetyrileasngluhisasnlysvalval380385390catttcaatctgcacatgcgcgccttagcgctgggacagagtgtgcgt1494hispheasnleuhismetargalaleualaleuglyglnservalarg395400405taccctatcaaatatattattaccgactttgatttattgcaaaagaac1542tyrproilelystyrileilethrasppheaspleuleuglnlysasn410415420tacgacataaagcaatatttagagagtaactttgatcttgtaataccg1590tyraspilelysglntyrleugluserasnpheaspleuvalilepro425430435440gaagagttgagacagaccgatctggttgcggacgtacgagtggtgtgc1638glugluleuargglnthraspleuvalalaaspvalargvalvalcys445450455gtcatcgacgacccatcacaaattataggtgaaccgtttagtttgtac1686valileaspaspproserglnileileglyglupropheserleutyr460465470gacggtaacgaacgagtttttgagagcacagtagccacggatggtaac1734aspglyasngluargvalphegluserthrvalalathraspglyasn475480485atgtatttagttggcgtgggtccgggagtgtacactctacgcgcgccc1782mettyrleuvalglyvalglyproglyvaltyrthrleuargalapro490495500cgcggcaaagacaaacgctacaaactccacttggcacactcgcccaac1830argglylysasplysargtyrlysleuhisleualahisserproasn505510515520gagccggttcatccggctaacgatcacatgtatctactcgtgacatat1878gluprovalhisproalaasnasphismettyrleuleuvalthrtyr525530535ccatattacaatcaaacgttaacctacacacgatatataacttcggac1926protyrtyrasnglnthrleuthrtyrthrargtyrilethrserasp540545550cttgcaatagacgcggcccacttattcggtaccgaccgcttgtatgtg1974leualaileaspalaalahisleupheglythraspargleutyrval555560565gccacgatatatttcgacgcattacagcagactgtgaccgtgtatctg2022alathriletyrpheaspalaleuglnglnthrvalthrvaltyrleu570575580aacaatattcgcactggcagggaaaacaacaccaccttgtattttgaa2070asnasnileargthrglyarggluasnasnthrthrleutyrpheglu585590595600atggaaatacataatccgtttattggcacttcttcgaaatttactttg2118metgluilehisasnpropheileglythrserserlysphethrleu605610615ttagaggataacgtcacgatgcgccagggatattataaatttccggcg2166leugluaspasnvalthrmetargglnglytyrtyrlyspheproala620625630gtcacctttagctcgattcgtttacacataagagatgacaacagacta2214valthrpheserserileargleuhisileargaspaspasnargleu635640645atgctggtagataaatatttaccagcgggcgacacgttgctgttcatg2262metleuvalasplystyrleuproalaglyaspthrleuleuphemet650655660tttcccaatcaaatcgttgacaataatatatttcccgatgggtcaata2310pheproasnglnilevalaspasnasnilepheproaspglyserile665670675680ttgaccagcacatacaaccgtataaaagaacaagctgctttcatcgaa2358leuthrserthrtyrasnargilelysgluglnalaalaph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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 902 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metsertyrasnvalilevalprothrthrvalleuproprotrpleu151015argileglyglnasntrpilephealaarghisargargthrgluval202530glyvalvalleuproalaasnthrlyspheargvalargalaaspphe354045alalystrpglyilethrargprovalilevalargleuleuasnasn505560asnargasnthrgluarggluileasnleuthrasnaspglntrpile65707580glumetgluhisgluhisglucysvalprophevalasptrpproval859095glyglulysasnthrmetalagluvalhisphegluileaspglypro100105110hisileproleuprovaltyrvalpheasnthrargprovalgluasn115120125phelysserglutyrargglnserserserglytyrcyspheleutyr130135140leuaspleuvalcysileleuvalproproalaserlysasnvalleu145150155160leuaspthraspleuphegluleuhisglnphetyrasngluileile165170175asntyrtyraspaspleucysglyleuvalgluaspprotyralaasp180185190thrvalaspserasnleuproasnlysalaalaphevallysalaasp195200205glyglyglyproglyglyalatyrtyrglyalaphetrpthralapro210215220alaserthrasnleuglyglutyrleuargvalserprothrasntrp225230235240metvalilehisgluleuglyhisalatyraspphevalphethrval245250255asnthrargleuilegluiletrpasnasnserphecysaspargile260265270glntyrthrtrpmetasnlysthrlysargglnglnleualaargile275280285tyrgluasnglnargproglnlysglualaalaileglnalaleuile290295300aspasnasnvalpropheaspasntrpaspphepheglulysleuser305310315320ilephealatrpleutyrasnproglnargglyleuaspthrleuarg325330335asnileasnhissertyrargleuhisalaalaargasnprovalthr340345350protyrproglniletrpalatrpleumetsercysglytyraspasn355360365phetrpleutyrpheasnargileglyleutyrproalaaspphetyr370375380ileasngluhisasnlysvalvalhispheasnleuhismetargala385390395400leualaleuglyglnservalargtyrproilelystyrileilethr405410415asppheaspleuleuglnlysasntyraspilelysglntyrleuglu420425430serasnpheaspleuvalileproglugluleuargglnthraspleu435440445valalaaspvalargvalvalcysvalileaspaspproserglnile450455460ileglyglupropheserleutyraspglyasngluargvalpheglu465470475480serthrvalalathraspglyasnmettyrleuvalglyvalglypro485490495glyvaltyrthrleuargalaproargglylysasplysargtyrlys500505510leuhisleualahisserproasngluprovalhisproalaasnasp515520525hismettyrleuleuvalthrtyrprotyrtyrasnglnthrleuthr530535540tyrthrargtyrilethrseraspleualaileaspalaalahisleu545550555560pheglythraspargleutyrvalalathriletyrpheaspalaleu565570575glnglnthrvalthrvaltyrleuasnasnileargthrglyargglu580585590asnasnthrthrleutyrpheglumetgluilehisasnpropheile595600605glythrserserlysphethrleuleugluaspasnvalthrmetarg610615620glnglytyrtyrlyspheproalavalthrpheserserileargleu625630635640hisileargaspaspasnargleumetleuvalasplystyrleupro645650655alaglyaspthrleuleuphemetpheproasnglnilevalaspasn660665670asnilepheproaspglyserileleuthrserthrtyrasnargile675680685lysgluglnalaalapheilegluasnhislysglnleuleutyrile690695700gluasngluleuargaspseriletyrleualaserglnphevalasn705710715720seraspserasnglupheleulystyrpheproasptyrpheargasp725730735prohisthrphesertyrleupheargpheargglyleuglyaspphe740745750metleuleugluleuglnilevalproileleuasnleualaserval755760765argvalglyasnhishisasnglyprohissertyrpheasnthrthr770775780tyrleuservalgluvalargaspthrserglyglyvalvalpheser785790795800tyrserargleuglyasngluprometthrhisgluhishislysphe805810815gluvalphelysasptyrthrilehisleupheileglngluprogly820825830glnargleuglnleuilevalasnlysthrleuaspthralaleupro835840845asnserglnasniletyralaargleuthralathrglnleuvalval850855860glygluglnserileileileseraspaspasnaspphevalpropro865870875880proproargvalasncysglyaspglnglnileargvalvalgluthr885890895leulysmetilealaphe900__________________________________________________________________________