Patent Abstract:
clones are disclosed comprising at least part of a dna sequence of a gene encoding maize acetyl coa carboxylase , or a sequence showing substantial homology therewith , flanked by heterologous dna . dna from such clones may be used to isolate similar sequences in other plants . such sequences may be used to transform plants to give a variety of useful effects , including resistance to herbicides such as fluazifop , and modified oil - bearing properties .

Detailed Description:
in the examples we describe the use of affinity purified antibodies to maize accase to select clones from a maize cdna expression library ; and we show that three of the clones obtained encode maize accase . confirmatory evidence for the identity of the clones is as follows : ( i ) amino acid sequences , deduced from dna sequencing of one of the clones , were found to be up to 50 % identical with parts of the rat and chicken accase sequence . ( ii ) the longest cloned cdna insert ( 4 . 5 kb ) is considerably longer than the predicted length of any mrna encoding known biotin - containing enzymes in plants , other than that encoding accase . we believe that the a3 and a4 clones represent two different maize accase genes . we have also found that the maize accase clones hybridise to southern blots of arabidopsis dna . this enables us to obtain from such arabidopsis dna probes suitable for isolation of acc - type coding sequences and genes from other plants , both dicots and monocots . plant material . seedlings of maize ( zea mays dekalb xl81 ) were grown in sterile soil , in a glasshouse with the temperature maintained between 20 ° c . and 30 ° c . under natural illumination . leaves of 2 to 4 week old plants were used for enzyme preparation . purification . extraction and purification procedures were carried out at 0 °- 4 ° c . extraction . leaf material ( 160 g ) was homogenised in a waring blender with 300 ml of medium consisting of 0 . 1m tris - hcl buffer ( ph 8 . 0 ), 10 mm mgcl 2 , 1 mm edta , 20 mm 2 - mercaptoethanol , 0 . 2 mm pmsf , 2 mm benzamidine hydrochloride , and 2 % ( w / v ) of polyvinylpolypyrrolidone . the homegenate was filtered through two layers of muslin and two layers of miracloth and the filtrate ( 380 ml ) centrifuged at 13000 g for 30 minutes . ammonium sulphate and polyethylene glycol fractionation steps . to the centrifuged extract ( 365 ml ) was added sufficient solid ammonium sulphate with stirring to bring the mixture to 40 % saturation . after 40 minutes gentle stirring the mixture was centrifuged at 13000 g for 30 minutes and the supernatant discarded . the protein pellet was re - dissolved in a medium consisting of 20 mm tris - hcl ( ph 8 . 0 ) 10 mm mgcl 2 , 1 mm edta , 10 mm dithiothreitol , 0 . 2 mm pmsf , and 10 % ( v / v ) glycerol . to this solution ( 204 ml ) was added solid peg ( 8 g per 100 ml ) with stirring . after 40 minutes gentle stirring the suspension was centrifuged as above and the pellet discarded . more peg was added to the supernatant ( 12 g per 100 ml ) and , after stirring for 40 minutes , the mixture was again centrifuged and the pellet kept . affinity chromatography on matrix gel orange a . the precipitated protein was re - dissolved in buffer consisting of 20 mm hepes koh ( ph 6 . 8 ), 10 mm mgcl 2 , 1 mm edta , 10 mm dtt , and 10 % ( v / v ) glycerol and clarified at 10000 g for 20 minutes . the supernatant ( 100 ml ) was passed through a 40 ml column of orange a gel ( previously equilibrated with column buffer consisting of the above dissolving medium but containing 20 mm 2 - mercaptoethanol instead of dtt ) at a flow rate of approximately 0 . 4 ml per minute . the column was then washed with 800 ml of column buffer at about 1 ml per minute to remove unbound protein . acetyl coa carboxylase was subsequently eluted from the column with 100 ml of 0 . 5 mm coa in dissolving buffer followed by more buffer . fractions containing acetyl coa carboxylase activity were pooled ( 100 ml ) and concentrated by ultra - filtration over a pm30 membrane ( amicon scientific ) to a volume of 5 . 8 ml . the enzyme solution was divided into aliquots and rapidly frozen ( in liquid nitrogen ) before storage at - 80 ° c . acetyl coa carboxylase activity was measured at 30 ° c . by the enzyme and substrate dependent incorporation of radioactivity from nah 14 co 3 into acid - stable products , based on published methods . reaction mixtures contained ( in a total volume of 200 μl ): 100 mm tris - hcl ( ph 8 . 0 ), 50 mm kcl , 5 mm mgcl 2 , 5 mm dtt , 2 mm atp , 15 mm nah 14 co 3 approx . 0 . 25 ci per mole ), 0 . 75 mm acetyl coa ( trilithium salt ), and enzyme ( 2 - 10 mu ). reactions were initiated with enzyme and stopped after 6 minutes by adding 50 μl of 6m hcl . portions of the reaction mixture were spotted into filter paper discs , which were then dried and the acid - stable 14 c reaction products measured by scintillation counting . the amount of enzyme which catalyses conversion of 1 μmol of substrate per min is defined as 1 unit . protein concentration was measured by coomassie blue dye binding according to published methods and using bovine serum albumin as standard . sds - page and electroblotting to nitro - cellulose or pvdf membranes were carried out according to published methods . 3 . 7 ml of a preparation of accase ( partially purified essentially by the above procedure ) containing about 680 μg of protein were concentrated to 100 μl using centrifugal filters ( amicon centricon 100 ). the concentrated protein solution was added to 80 μl of a digestion mixture containing 125 mm tris - hcl ( ph 6 . 7 ), 2 % ( w / v ) sds , 10 % ( w / v ) glycerol and 0 . 01 % ( w / v ) bromophenol blue , and the solution heated at 98 ° c . for 5 minutes . the digest was applied to several tracks of a 7 . 5 % polyacrylamide gel and the protein subunits separated by sds - page . the gel was subsequently washed with 5 mm tris - hcl ( ph 6 . 7 ) then stained with coomassie blue g ( 1 % w / v in water ) before destaining with water . segments of the stained gel containing accase subunit were cut out . gel segments containing accase subunit were gently agitated in 4 ml of uha ( urea / water / acetic acid ; 25 g : 25 ml : 25 ml ) for 80 min with one change of solution . the segments were then treated with 4 ml of a solution of 0 . 2 % ( w / v ) n - chlorosuccinimide in uha for 60 minutes to cleave the accase subunit at tryptophan residues , followed by washing with 5 mm tris hcl , ph 6 . 7 ( 30 min ). the gel segments were then equilibrated with a solution containing 125 mm tris - hcl ( ph 6 . 7 ), 5 % ( w / v ) peg , 20 mm dtt , 1 % ( w / v ) sds , for 90 min , finally heating the segments in solution to 95 ° c . for 6 min . the gel segments , containing cleaved fragments of accase subunit , were then loaded onto several tracks of a 16 % acrylamide gel and the fragments separated by sds - page . after separation , the polypeptide fragments were electroblotted onto a pvdf membrane by published methods and the membrane stained with amido black . portions of the membrane containing major polypeptide fragments were excised and destained exhaustively with 10 mm tris base , then washed with water before being submitted for n - terminal amino acid sequencing . n - terminal amino acid sequencing was carried out by established techniques at the biomolecular resources facility of the australian national university . orange a - purified maize accase (˜ 50 μg protein in 1 ml freund &# 39 ; s complete adjuvant ) was injected into the hind leg muscle of a rabbit . four weeks later a further 50 μg of the maize accase in incomplete freund &# 39 ; s adjuvant was similarly injected . the rabbit was bled at two to three week intervals and serum collected . the first serum sample did not contain detectable antibodies to maize accase as judged by binding to maize accase after sds - page and immunoblotting or inhibition of maize accase activity . the second and subsequent bleedings yielded antiserum that bound to maize accase as judged by the two criteria listed above . this antiserum was used as the starting material for purification of affinity - purified antibody . partially purified maize accase ( 500 μl ) was resolved from contaminants by preparative sds polyacrylamide gel electrophoresis . the membranes were stained with amido black and the accase band at ˜ 200 kda was cut out . residual adsorption sites on the membrane were blocked by incubation of the membrane in tbs ( 20 mm tris - kcl , ph 7 . 5 , 150 mm nacl ) containing 5 % ( w / v ) powdered milk . antibodies to maize accase were adsorbed to the membrane by incubating the accase - coated membrane in 1 ml dilute antiserum ( 0 . 5 ml antiserum 0 . 5 ml tbs ) at room temperature for 1 hour . the membrane was removed , washed for 5 minutes in 10 ml tbs + 0 . 05 % tween 20 then twice ( 5 min each ) in 10 ml tbs . the membrane - bound antibodies were eluted by incubation with 1 ml 0 . 1m glycine / hcl ph2 . 6 for 3 minutes and neutralised . this cycle of adsorption and elution was repeated two more times using the same membrane and antiserum . before use the affinity - purified antibody was diluted to 20 ml with tbs . this antibody preparation was further depleted of non - specific antibodies by incubation with nitro - cellulose coated with an e . coli λ phage lysate . finally , the affinity - purified antibody was made 10 % with respect to horse serum albumin to further minimise non - specific binding . a series of dilutions of serum from control and accase - immunised rabbits were prepared . enzyme extract ( 50 μl ) supplemented with 0 . 02 % ( v / v ) triton x - 100 was mixed with diluted serum ( 50 μl ) and incubated at 30 ° c . for maize , the enzyme was orange - a purified while for amaranthus edulis a g - 25 treated crude leaf extract prepared from 1 g of tissue in 2 ml of solution containing 0 . 1m tris - hcl ( ph 8 . 0 ), 10 mm mgcl 2 , 1 mm edta , 10 mm dtt , 0 . 01 % ( v / v ) triton x - 100 was used . after 50 minutes incubation of serum with maize enzyme , immune complexes were removed by adding 20 μl of a suspension of protein a - sepharose ( 0 . 2 g per ml in 0 . 1m kh 2 po 4 buffer ph 7 . 0 ) and centrifuging ( 11600 g , 5 min ) after a further 60 min at room temperature . enzyme activity remaining in the supernatant was measured . incubation of amaranthus edulis extract with serum was for 160 min before removal of immunoprecipitates with protein a and centrifugation ( 5 min , 11500 g ). the a . endulis mixtures were left a further 16 hours at 4 ° c . and centrifuged again before assay of remaining enzyme in the supernatant . this was a gift from dr a barkan , department of botany , university of california , berkeley . messenger rna was isolated from leaves of two week old zea mays ( b73 ) seedlings . cdna was synthesised using a zap cdna synthesis kit ( stratagene ). the cdna was ligated into the ecori site ( 5 &# 39 ; end of the cdna ) and the xhoi site ( 3 &# 39 ; end of the cdna ) of lambda expression vector λzap . the library contained 8 × 10 5 recombinants . the maize λzap cdna library was screened for clones producing fusion proteins that would bind to antibodies purified by adsorption to purified maize accase . phage was adsorbed to e . coli y1090 cells mixed with 0 . 8 % agarose / 10 mm mgso 4 / 0 . 02 % maltose in lb , plated at a density of about 100 plaques cm 2 . the plates were incubated at 37 ° c . for about 4 hours ; then nitro - cellulose filters impregnated with iptg were placed on the agarose surface and the incubation continued at 37 ° c . overnight . the filters were removed , washed in tbs buffer containing 5 % ( w / v ) powdered milk to block the surface of the nitro - cellulose before screening with affinity purified antibody . the filters ( 6 × 137 mm diam ) were incubated in 20 ml of the affinity purified antibody for 2 hours at room temperature . the filters were washed in tbs containing 0 . 05 % tween 20 three times , then in goat anti - rabbit immunoglobulin coupled to alkaline phosphatase ( 1 : 7500 in ttbs , 0 . 2 % tween 20 in tbs ) for 1 hour at room temperature . the filters were freed of unbound 2nd antibody by washing twice in ttbs then once in tbs . the plaques expressing accase - fusion proteins were visualised by incubating on an alkaline phosphatase reaction mixture containing bcpip and nbt ( 150 μl / ml bcpip , 150 μl / ml nbt in 100 mm tris - hcl , ph 9 . 5 , 100 mm nacl , 1 mm mgcl 2 ). plaques giving a positive reaction were further purified by repeated rounds of screening using the same antibody solution until all plaques on a plate gave a positive signal . three positive plaques were obtained by this procedure . following the procedure detailed above , accase was purified over 100 - fold from maize leaves to a specific activity of 3 - 4 units per mg protein ( table 1 ). analysis by sds - page of the enzyme at different stages of purification showed that accase consisted of polypeptide subunits of about 200 - 220 kda and , at the final stage , was about 70 % pure ( fig1 ). the presence of biotin in the accase subunit was demonstrated by western blotting of the sds - polyacrylamide gel and assaying with streptavidin - phosphatase . a number of degradation products of accase were also shown by this sensitive procedure . attempts to determine the n - terminal amino acid sequence were unsuccessful , presumably because it was blocked . however , limited internal sequence data has been by cleaving the electrophoretically purified subunit with n - chlorosuccinimide , re - electrophoresing the products and electroblotting to pvdf membrane for n - terminal sequence analysis . the three sequences obtained were 8 , 4 and 8 amino acids in length and were 62 . 5 %, 100 % and 75 % similar to sequences in chicken accase ( table 2 ). orange - a purified accase was injected into a rabbit and , following two booster injections , the rabbit serum was found to form immune complexes with maize accase activity . accase activity from amaranthus was also immunoprecipitated by similar concentrations of antiserum . accase antibodies were affinity purified by adsorption to and elution from a pvdf membrane containing electrophoretically purified accase subunit . this purified antibody preparation was used to screen the maize cdna expression library . approximately 90 , 000 plaques of a maize cdna expression library constructed in the vector λzap ( strategene ) were screened with affinity - purified accase antibody . of the seven clones picked in the primary screening , three remained positive through two further rounds of purification . two of these ( a1 and a3 ) gave a much stronger signal than the third ( a4 ). analysis of the bluescript phagemids released from the λzap clones indicated they were carrying dna inserts of 4 . 0 ( a1 ), 4 . 4 ( a3 ) and 4 . 4 ( a4 ) kb . restriction mapping showed the 4 . 0 and 4 . 4 kb inserts of a1 and a3 to be almost identical , except for the extra 400 bp at the 5 &# 39 ; end . the map of a4 was different but the a4 insert cross - hybridised to a1 / a3 . re - screening of the λzap library with the a3 insert yielded a further six positively hybridising clones . restriction mapping established that four of these were like a1 / a3 and two were like a4 . fig2 shows the digestion pattern for an a4 - like cdna ( a12 ) and an a3 - like cdna ( a34 ) cut with a number of different restriction enzymes . these results indicate there are at least two genes for accase in maize . the dna sequence of the a3 acc cdna clone was determined ( fig3 ). the deduced amino acid sequence was 37 % identical ( 58 % similar ) to the rat / chicken accase sequence and , over one stretch of 100 residues , the sequences were more than 60 % identical . the sequence of most of the a1 acc cdna was determined and found to be identical to the a3 sequence . additional sequence information for the 5 &# 39 ; ends of longer a3 - like clones are given in fig4 ( a , b ). in the case of clone a10 / a49 , the sequence includes the motif that encodes the conserved biotin - binding site , met - lys - met ( fig4 b ) of accase . analysis of the 5 &# 39 ; ends of two a4 - like clones , a12 and a4 acc cdnas ( fig5 a , b ), and of the 3 &# 39 ; end of a12 ( fig5 c ) showed there are a significant number of differences between the sequences of a3 - and a4 - like cdnas . this supports the conclusion drawn from the restriction digest patterns that there must be at least two accase genes in maize . hybridisation of the a3 cdna to southern blots of ecor1 and bamh1 digested maize dna gave 3 and 2 bands respectively . it is likely therefore that there are no more than two accase genes in maize . the maize cdna also hybridised to arabidopsis dna under low stringency conditions . hybridisation of the a3 cdna insert to a northern blot of maize rna gave just one band of size 8 . 0 - 8 . 5 kb . the size of the maize accase subunit (˜ 220 kda ) is similar to that of the enzyme from rat and chicken . the mrna for such a protein would be expected to be at least 6 . 5 kb and according to our determination is in fact 8 . 0 - 8 . 5 kb for maize . this is considerably longer than the longest cdnas ( 5 . 5 kb for a3 - like and 5 . 7 kb for a4 - like clones ) described here . these cdnas , however , will provide the means for isolating full - length cdnas . the cdnas may be used as primers for 5 &# 39 ; extension against maize mrna as a template or as probes for isolating the genes from a maize genomic library . the maize accase cdna cross - hybridised to arabidopsis dna and it therefore presents a means for isolating the gene from an arabidopsis genomic library . the same heterologous probe may be used for isolating accase genes from a range of plants , including both monocots and dicots , for example oil - seed rape . there are clearly two accase genes in maize . as only one band is observed when a northern blot of maize leaf rna is hybridised with the a3 cdna it would seem the messenger rnas for both genes are the same size . more a3 - like than a4 - like clones were selected from the maize leaf cdna library so the a3 mrna must be the more abundant . of the three cdna clones initially selected from the maize expression library by their reaction with antibodies to maize accase , a1 and a3 gave the strongest signal with the antibody and a4 the weakest . this could indicate that the a3 - like cdnas encode the grass - weed herbicide - sensitive form of accase . whether the enzyme encoded by the a4 - like cdna is herbicide - sensitive or tolerant is unclear . by defining the 5 &# 39 ; end of a full - length maize cdna it will be possible to isolate the promoter sequence from a maize genomic clone . similarly it will be possible to isolate the promoter for the arabidopsis gene and by reciprocal constructions to express the herbicide - tolerant arabidopsis enzyme in herbicide - sensitive monocot species ( for example , maize , wheat , barley , rice ). this provides a method for producing monocotyledonous crop plants tolerant to herbicides ( such as fluazifop ) that are active by interfering with the fatty acid biosynthesis pathway . finally , the invention enables the use of a full - length cdna clone to synthesise active accase in a transformed micro - organism culture , such as an e . coli or yeast system . such a system would be useful for selecting herbicide - resistant forms of the accase enzyme , and for testing new compounds for herbicidal activity . it would also allow investigation of the nature of the herbicide - binding site and of the basis of at least one of the mechanisms responsible for resistance to grass - weed herbicides . note : fig2 shows restriction digests of the a4 - like ( acc12 ) and the a3 - like ( acc34 ) maize accase cdna clones . the oddnumbered lanes show acc12 and the even - numbered ones show acc34 . the enzymes used are pst1 ( 1 , 2 ), bamh1 + bg1ii ( 3 , 4 ), hindiii ( 5 , 6 ), hincii ( 7 , 8 ), ecor1 ( 9 , 10 ), acci ( 11 , 12 ), pvuii ( 13 , 14 ), pvuii + hincii ( 15 , 16 ), pvuii + ecorv ( 17 , 18 ). marker lanes ( m ) are λ dna digested with hindiii . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 11 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 4345 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1313 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 400 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 3 : caaatacacaattgaatctgtaaggactggacatggtagctacaggttgagagtgaatga60ttcaacagttgaagcgaatgtacaatctttatgtgatggtggcctcttaatgcagttgga120tggaaacagccatgtaatttatgcagaagaagaagctggtggtacacggcttcagattga180tggaaagacatgtttattgcagaatgaccatgatccatcaaagttattagctgagacacc240ctgcaaacttcttcgtttcttggttgctgatggtgctcatgttgatgcggatgtaccata300cgcggaagttgaggttatgaagatgtgcatgcctctcttgtcacctgcttctggtgtcat360tcattgtatgatgtctgagggccaggcattgcaggctggt400 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 133 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 4 : lystyrthrilegluservalargthrglyhisglysertyrargleu151015argvalasnaspserthrvalglualaasnvalglnserleucysasp202530glyglyleuleumetglnleuaspglyasnserhisvaliletyrala354045glugluglualaglyglythrargleuglnileaspglylysthrcys505560leuleuglnasnasphisaspproserlysleuleualagluthrpro65707580cyslysleuleuargpheleuvalalaaspglyalahisvalaspala859095aspvalprotyralagluvalgluvalmetlysmetcysmetproleu100105110leuserproalaserglyvalilehiscysmetmetsergluglygln115120125alaleuglnalagly130 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 230 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 5 : gttgaatttttaccatggaaaaaacgaggactttccatccaagttgctaagagacatcat60tgaggaaaatctttcttatggttcagagaaggaaaaggctacaaatgagaggcttgttga120gcctcttatgaacctactgaagtcatatgagggtgggagagagagccatgcacattttgt180tgtcaagtctcttttcgaggagtatcttacagtggaagaactttttagtg230 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 76 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 6 : leuasnphetyrhisglylysasngluasppheproserlysleuleu151015argaspileileglugluasnleusertyrglyserglulysglulys202530alathrasngluargleuvalgluproleumetasnleuleulysser354045tyrgluglyglyarggluserhisalahisphevalvallysserleu505560phegluglutyrleuthrvalglugluleupheser657075 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 250 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 7 : gttcaagctgagaggcccccatggtatatctcagtggttggaggtgctttatataaaaca60gtaaccaccaatgcagccactgtttctgaatatgttagttatctcaccaaaggccagatt120ccaccaaagcatatatcccttgtcaattcaacagttaatttgaatatagaagggagcaaa180tacacaattgaaactgtcaggactgggcatggtaggtacaagttgcgaatgaatgattca240acagttgagg250 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 83 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 8 : valglnalagluargproprotrptyrileservalvalglyglyala151015leutyrlysthrvalthrthrasnalaalathrvalserglutyrval202530sertyrleuthrlysglyglnileproprolyshisileserleuval354045asnserthrvalasnleuasnilegluglyserlystyrthrileglu505560thrvalargthrglyhisglyargtyrlysleuargmetasnaspser65707580thrvalglu ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 330 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 9 : tatggttcagagaaggaaaaggctacaaatgagcgacttgttgagcctcttatgaaccta60ctgaagtcatatgagggtgggagagaaagccatgcacattttgttgtcaagtctcttttt120gaggagtatcttaccgtggaagaacttttcagtgatggcattcagtctgacgtgattgaa180accttgcgtcatcagcacagtaaagacctgcagaaggttgtagacatcgtgctgtctcac240cagggtgtgaggaacaaagctaagcttgtaacagcacttatggaaaagctggtttatcca300aatcctgcattgcaagaagcctttcagatc330 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 110 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 10 : tyrglyserglulysglulysalathrasngluargleuvalglupro151015leumetasnleuleulyssertyrgluglyglyarggluserhisala202530hisphevalvallysserleuphegluglutyrleuthrvalgluglu354045leupheseraspglyileglnseraspvalilegluthrleuarghis505560glnhisserlysaspleuglnlysvalvalaspilevalleuserhis65707580glnglyvalargasnlysalalysleuvalthralaleumetglulys859095leuvaltyrproasnproalaleuglnglualapheglnile100105110 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 319 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( xi ) sequence description : seq id no : 11 : tgcttgccccttgattgaccatgtctgatcctaagtcgaccattatttccttgaaacttc60ctttcggacgtggtgctatggttgatgaatttggatgtgtgcgttctgccaggtgtaagc120ccaaaggtttatacagaccgagttaaggttaggaagagcacgagtgaacctgttctggtt180ttgcagtggttaaggcagaaagttgtttcactgtagttctgagatgtattaccagcggcg240ctgtaattttagggtgtataatgcggatgctagtaaacaattgagtggttctttaaaaaa300aaaaaaaaaaaaaaaaaaa319__________________________________________________________________________