Patent Application: US-79382497-A

Abstract:
the present invention relates to plants genetically modified to increase the level of stored carbohydrates in the plant , particularly during periods of high sink activity and low source activity through production of a glycosyl - transferase which catalyzes the formation of soluble glucans . the invention also relates to the genetic constructs used to produce the engineered plants and the method of producing the engineered plants .

Description:
the invention - is further described with reference to the accompanying example which is no way limiting on the scope of the present invention . the general strategy adopted to isolate a gene from s . salivarius encoding a gtf which produces a primer independent and soluble glucan is as follows : a λ gene bank containing s . salivarius dna was prepared . positive clones were detected by using an e . coli strain grown on agar containing sucrose . e . coli which contained gtf dna from s . salivarius could convert the sucrose in the medium into a polymer which resulted in opaque colonies . these opaque colonies were then picked and the s . salivarius dna excised and subjected to restriction mapping to ascertain whether the dna was from a previously described s . salivarius gtf gene , or whether the dna was novel . three clones containing novel dna were located . these were subjected to a radioactive assay to determine whether the dna encoded for a primer independent or primer dependent gtf . one clone - λc - 13 was found to contain a novel gtf gene which coded for a primer independent gtf . the dna from this clone was then isolated and sequenced . escherichia coli le392 and nm522 and s . salivarius atcc 25975 were used . e . coli strains were grown in luria - bertani ( lb ) medium at 37 ° c ., supplemented with ampicillin ( 100 μg ml - 1 ), isopropylthiogalactoside ( iptg ) ( 1 mm ), or 5 - bromo - 4 - chloro - 3 - indolyl - β - d - galactoside ( x - gal ) ( 100 μg ml - 1 ) as appropriate . cultures of s . salivarius were grown at 37 ° c . in semi - defined medium ( sdm ) containing 25 mm glucose supplemented with 0 . 005μl tween 80 ml - 1 where appropriate . bacteriophage and phagemids . all genetic constructs , excluding sequencing subclones , are listed in table 1 . bacteriophage - λ derivatives were grown either as 20 ml or 1 l - liquid lysates using e . coli strain le392 as the host and dna purified according to the method of silhavy et al ( 1984 ). plasmids were propagated in e . coli strains as described previously ( giffard et al , 1991 ). screening of gene bank . a bacteriophage - λ gene bank of s . salivarius atcc 25975 ( pitty et al , 1989 ) was screened by detecting plaques on a lawn of e . coli le392 grown at 37 ° c . on minimal agar medium containing 0 . 2 % glucose and 50 μg ml - 1 methionine as well as it ( wt / vol ) sucrose with or without 0 . 02 % ( wt / vol ) dextran t - 10 . potential gtf clones were detected by their opacity including λc - 13 containing the gtf m gene . twenty recombinant plaques were picked from minimal media plates containing sucrose and the ecor1 restriction patterns of these recombinants were analysed . of these recombinants , only λc - 13 exhibited a unique ecor1 restriction pattern and gtf activity . a restriction map of λc - 13 was constructed using double restriction digests . the gtf gene encoded by λc - 13 ( gtfm ) was located on an 8 . 3 kbp insert ( see fig1 ). the 5 . 3 kbp xbai fragment from λc - 13 was subcloned into pibi31 30 ( pgsg501 ; see table 1 ) and was positive for gtf activity as was the 4 . 8 kbp xbai / ecor1 from λc - 13 subcloned into pibi31 ( pgsg502 ; see table 1 ). table 1__________________________________________________________________________bacterial strains , phages and phagemidsbacteria , phage or phagemid description source or reference__________________________________________________________________________bacterium : streptococcus salivarius atcc 25975 atcc ( hamilton , 1967 ) escherichia coli le392 f . sup .- e14 . sup .- ( mera . sup .-) hsdr514 ( r . sub . k . sup .- m . sub . k . sup .-) supe44 and supf58 murray et al ., 1977 lacy1 or δ ( lacizy ) 6 galk2 gal722 metb1 trpr55 escherichia coli nm522 f &# 39 ; laci . sup . 4 δ ( lacz ) m15 proa . sup .+ b . sup .+ / supe thi δ ( lac - proab ) gough and murray , 1983 δhsdms - mcrb ) 5 ( r . sub . k . sup .- m . sub . k . sup .- mcrbc . sup .-) bacteriophage : 47 . 1 loenen and brammar , 19808 47 . 1 with gtfj encoding 8 . 5 kbp sau3a partial fragment pitty et al ., 1989 of s . salivarius atcc 2597533 47 . 1 with gtfk encoding 9 . 6 kbp sau3a partial fragment pitty et al ., 1989 of s . salivarius atcc 2597513 47 . 1 with 8 . 3 kbp gtfm encoding sau3a partial fragment this study of s . salivarius atcc 2597510 47 . 1 with 11 kbp gtfl encoding sau3a partial fragment this study of s . salivarius atcc 2597540 47 . 1 with sau3a partial fragment of s . salivarius atcc 25975 thisstudy isolated from sucrose - containing medium phagemid : pibi30 ap . sup . r , f1 origin replication , β - galactosidase , ibicorporation t3 and t7 polymerase promoters pibi31 ap . sup . r , f1 origin replication , β - galactosidase , ibicorporation t3 and t7 polymerase promoters pgsg101 ( pgs101 ) pibi30 with gtfj encoding 6 . 8 kbp saci / bamiii fragmentof - 8 giffard et al ., 1991 pgsg201 ( pgs201 ) pibi30 with gtfk encoding 7 . 3 kbp bg / ii / bamiii fragmentof - 33 giffard et al ., 1991 pgsg401 pibi30 with gtfl encoding 6 . 2 kbp bamiii / xbai fragment of - 10this study pgsg402 pibi31 with 6 . 2 kbp bamiii / xbai fragment of - 10 this study pgsg403 pibi30 with 4 . 8 kbp ecori fragment of - 10 this study pgsg404 pibi30 with 4 . 1 kbp ecori fragment of - 10 this study pgsg502 pibi31 with gtfm encoding 5 . 3 kbp xbai fragment of - 13 thisstudy pgsg502 pibi31 with gtfm encoding 4 . 8 kbp ecori / xbai fragment of - 13this study pgsg503 pibi31 with 3 . 7 kbp kpni / xbai fragment of - 13 this study__________________________________________________________________________ detection of gtf activity . gtf activity was routinely detected using a qualitative microtitre reducing sugar test for liberated fructose , outlined in jacques n . a . ( 1983 ). gtf activity encoded by phagemids was released from e . coli cells by permeabalizing 1 ml of a stationary phase culture . this was achieved by vortexing the cells in the presence of 50 μl 0 . 1 % ( wt / vol ) sds and 100 μl chloroform for 20 seconds . quantification of gtf activity utilized [ u - glucosyl - 14 c ]- labelled sucrose . one unit of enzyme activity was defined as the amount of gtf that catalyzed the incorporation of 1 μmol of the glucose moiety of sucrose in 75 % ( vol / vol ) ethanol - insoluble polysaccharide per min . the assay mix used for the quantification of gtf activity was scaled up to 8 ml and incubated with 3 . 2 ml of bacteriophage λ lysates at 37 ° c . for 2 h . after the 2 h incubation , the assay mix was boiled for a further 1 h to inactivate the enzyme and the amount of glucan formed ( cpm ) determined by assaying duplicate 500μl aliquots . after cooling to 37 ° c ., c . gracile endo -( 1 → 6 )- α - d - glucanase was added to a final concentration of 500 mu / ml and the solution incubated at 37 ° c . duplicate aliquots ( 500μl ) were removed and assayed for total remaining glucan at varying time intervals over a 5 h period . any reduction in glucan ( cpm ) during this period was attributed to hydrolysis by the endo -( 1 → 6 )- α - d - glucanase . dna sequence analysis . dna sequence determination was carried out on cscl purified double - stranded dna using the pharmacia t7 sequencing kit according to the manufacturer &# 39 ; s instructions . custom - made oligonucleotide primers ( 17 mers ) were used and all sequencing was confirmed in both directions . dna sequences were assembled and open reading frames ( orfs ) detected using the ibi - pustell sequence analysis software version 2 . 03 . southern hybridizations . chromosomal dna from s . salivarius atcc 25975 was extracted and purified as previously described ( giffard et al , 1991 ). southern hybridizations were done essentially as outlined by silhavy et al ( 1984 ) and in accordance with standard techniques such as those described in maniatis et al ( 1989 ). incorporation into plants . incorporation of gtfm gene into plants is obtained by standard transgenic techniques . the gtfm gene is obtained from λc - 13 or pgsg501 by pcr . various constructs are made using pcr primers that either do or do not contain a coding region that adds a vacuolar targeting sequence to the n - or c - terminus of the gtfm protein . these pcr constructs are cloned into a puc18 based vector containing a cauliflower mosaic virus ( camv ) 35s promoter . by this means the streptococcal promoter is replaced by a plant promoter . other methods of incorporating foreign dna into plants are taught in australian patent application no . 46881 / 89 by ciba geigy ag . they include the use of agrobacterium tumefaciens and the leaf disc transformation method and the use of tobacco mosaic virus ( tmv ). __________________________________________________________________________ # sequence listing - - - - ( 1 ) general information : - - ( iii ) number of sequences : 2 - - - - ( 2 ) information for seq id no : 1 : - - ( i ) sequence characteristics : ( a ) length : 4853 base - # pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - - ( ii ) molecule type : dna ( genomic ) - - ( iii ) hypothetical : no - - ( iv ) anti - sense : no - - ( vi ) original source : ( a ) organism : streptococcu - # s salivarius - - ( xi ) sequence description : seq id no : 1 : - - cagagattta tgaaaagaag atgatttttt cctatttgta atttgtctga at -# atatcata 60 - - gagtagagat gacaacagaa aaaaggatga ttgatataga tggaaaataa gg -# tacgtttt 120 - - aaactacaca aggttaagaa aaactgggta actattgggg tgaccactct ct -# caatggtt 180 - - gccttggcag gtggaagcct cctagctcaa ggaaaagtag aggccgatga ga -# cgagcgca 240 - - cctaacggtg acggcttgca gcaactgagt gaggatggga ctgccagtct ag -# tgacgaca 300 - - acaactgtta ctgagcaagc tagtgctcaa gcaagtgtgt cagcagtagc aa -# cagccagc 360 - - gtaagtcacg aaacaagctt ccaggcggcg acaagtgcag tcagccagga gg -# caactgct 420 - - caagcacaaa ctagtccagt tgccagtcaa gaagtggcag tatcttcgca aa -# ctcaatcc 480 - - agtggccaag agacacagac tactgaacag gtgtcacaag gtcagacatc aa -# ctcaagta 540 - - gctgggcaaa caagtgctca gtctactcca agtgtgacag aacaagcaag ac -# ctagagtc 600 - - ttgaccaatg cagcgccagc aattgccaca cgcgctgctg atagcactat tc -# gtatcaat 660 - - gccaaccgca atactaacat cacgattacg gccagcggta cgacaccaaa tg -# taaccatt 720 - - atcacagggc caaacacgcc taaaccaaac gtgacggtga caagtccaaa tg -# gcacaaga 780 - - ccaaatgtga ccattgtaac gcagccaaat caacccaaca aacctgttca ac -# caagtcaa 840 - - ccgtctcaac ctaacaagcc ggtccaacca aatcagccaa gtcttgacta ta -# aaccagta 900 - - gcctctaact tgaagactat cgatggcaag cagtactatg ttgaaaatgg cg -# tcgtgaaa 960 - - aagaacgcag ccattgagct tgatgggcgt ctttattatt tcgatgagac tg -# gagctatg 1020 - - gtggatcaaa gtaaaccttt gtatcgtgcc gatgccattc caaataactc ta -# tctatgcg 1080 - - gtttataacc aggcctatga tacgtcaagt aagagttttg aacacttgga ta -# atttcttg 1140 - - acggctgata gctggtaccg tccaaaacag attttgaagg atgggaagaa tt -# ggacagct 1200 - - tcaactgaga aagattatcg tccacttttg atgacttggt ggccagacaa gg -# tgacacag 1260 - - gtcaactacc tcaactatat gagccaacaa gggtttggta ataagaccta ta -# cgacagat 1320 - - atgatgagct atgacttggc agctgctgcc gaaacggttc aacgaggcat cg -# aggaacgt 1380 - - attggtcgtg agggcaatac cacttggctt cgccagttga tgtcagactt ca -# tcaaaaca 1440 - - cagcctggct ggaactctga gagtgaggac aatctcttag ttggtaaaga cc -# acttgcaa 1500 - - ggaggcgctc tgaccttcct aaataatagt gcgacaagtc atgccaattc ag -# atttccgc 1560 - - ctcatgaacc gcacaccgac taaccaaacg gggacacgca aatatcatat tg -# accgttca 1620 - - aatggtggtt acgagttgct cttggctaac gatatcgaca actctaaccc ag -# ctgttcag 1680 - - gcagagcaac taaactggct ccactacatc atgaacattg gttctatcct tg -# gcaatgat 1740 - - ccaagtgcca actttgacgg tgttcgtatc gatgcggtgg acaatgtgga tg -# cggatctc 1800 - - ttgcagattg cttctgatta cttcaaggaa aaataccgtg tcgcagataa tg -# aagcaaat 1860 - - gccattgctc atttgtcaat ccttgaagct tggtcataca acgaccacca at -# acaacaag 1920 - - gataccaagg gtgctcagtt gtctatcgac aatccactac gtgaaacgct tt -# tgacgact 1980 - - ttcttgcgta agagcaatta ccgtggcagc ttggagcgcg tgattactaa ct -# ctcttaac 2040 - - aatcgttcaa gtgagcagaa acacacgcca cgtgatgcca attatatttt cg -# tgcgagcc 2100 - - catgatagtg aagtgcaagc tgttttggct aatatcatca gcaagcagat ta -# atccaaaa 2160 - - acagatggtt tcaccttcac tatggatgag ctcaaacagg ccttcgaaat ct -# acaatgcg 2220 - - gacatcgcga aggctgataa aaagtacacc cagtacaata tcccagctgc ct -# atgccaca 2280 - - atgttgacca acaaggatag tatcactcgt gtttactacg gggacctctt ta -# ccgacgat 2340 - - ggccaataca tggctgaaaa atcaccgtac tataatgcca tcgatgccct gc -# tccgtgct 2400 - - cgcatcaaat atgtagcagg tggtcaagac atgaaggtta ctaagcttaa tg -# gctatgaa 2460 - - atcatgtcat ctgtgcgtta tggtaagggg gcagaagaag ccaaccagct tg -# gtactgct 2520 - - gaaacacgca accaaggaat gctggtcctt acagccaacc gtccagatat ga -# agttggga 2580 - - gctaatgatc gtctggtagt caatatggga gctgcccaca aaaatcaggc tt -# accgtcca 2640 - - ttgcttctca gcaaatcgac aggtcttgcg acctacctca aggattctga tg -# taccagct 2700 - - ggattggttc gctatacgga caatcaaggg aacttgacct tcacggcaga tg -# atattgct 2760 - - ggtcattcaa ccgttgaagt ttcaggttat ttggcagttt gggtgccagt gg -# gtgcctca 2820 - - gaaaaccaag atgcccgaac caaggcttcg agcaccaaga agggtgagca gg -# tctttgaa 2880 - - tcatcagcag ctcttgattc acaagtcatt tacgaaggct tctcaaactt cc -# aagatttc 2940 - - gttaagacac caagtcagta caccaaccgt gttattgctc aaaatgccaa ac -# tcttcaaa 3000 - - gagtggggaa tcacttcctt tgaatttgcg ccacagtatg tgtctagcca ag -# acggcact 3060 - - ttcttggatt ctatcattga aaatggctac gccttcgagg accgctacga ta -# ttgccatg 3120 - - agcaagaaca acaaatacgg ttcactcaaa gacctcatgg atgcccttcg tg -# cccttcac 3180 - - gcagaaggta tctcagccat tgctgactgg gttccagatc aaatttacaa tc -# tccctgga 3240 - - aaagaagtgg taacagcttc tcgtaccaat agctacggta caccacgtcc aa -# atgctgaa 3300 - - atctacaata gcctctacgc agctaaaaca cgtacctttg gaaatgactt cc -# aaggcaag 3360 - - tacggtggtg ccttccttga tgaattgaag gcaaaatacc cagcaatctt tg -# agcgcgtg 3420 - - cagatttcaa acggccgtaa attgactacc aatgagaaaa tcacgcaatg gt -# cagccaag 3480 - - tatttcaatg gaagcaatat ccaaggtact ggagctcgct atgtcctaca ag -# ataacgct 3540 - - accaaccaat acttcagcgt caaagcaggt caaaccttcc ttcctaaaca aa -# tgactgaa 3600 - - attactggaa gtggtttccg tagggttgga gatgatgtcc aatacctctc aa -# ttggtggc 3660 - - taccttgcta agaatacctt tattcaagtc ggtgccaacc agtggtatta ct -# ttgataag 3720 - - aatggcaaca tggtcacagg tgagcaggtc attgatggca agaaatactt ct -# tcctagac 3780 - - aatggtctcc agctacgtca tgtccttcgc caaggtagtg atggtcatgt gt -# attattac 3840 - - gatcctaaag gggttcaggc ctttaacgga ttttatgatt ttgcgggtcc tc -# gccaagac 3900 - - gttcgctact ttgatggcaa cggtcaaatg tatcgtggcc tccacgacat gt -# atggcaca 3960 - - accttctatt ttgatgaaaa gactggtatt caagccaaag acaagttcat cc -# gttttgcg 4020 - - gacggacgca cgcgttactt catcccagat acaggaaatc tcgcagtcaa cc -# gatttgcg 4080 - - caaaatcctg agaacaaggc ttggtattac ctcgatagca acggttatgc cg -# tgacagga 4140 - - ctacaaacca ttaacggtaa gcagtattac tttgacaatg aaggacgtca gg -# ttaaggga 4200 - - cactttgtca ctatcaataa ccaacgttac ttccttgatg gtgatagtgg tg -# aaattgct 4260 - - ccgtcacgct ttgtgacgga aaacaacaag tggtactatg tcgatggcaa tg -# gtaaactg 4320 - - gttaaaggtg ctcaggtcat caatggtaat cactactatt tcaacaatga tt -# atagccaa 4380 - - gtcaagggtg cctgggccaa cggccgttac tatgatggtg actcaggtca gg -# ccgtaagc 4440 - - aaccaattca ttcaaattgc ggctaaccaa tgggcttacc ttaaccaaga tg -# gtcacaaa 4500 - - gtaacaggtc ttcaaaatat taacaataaa gtttactatt ttggtagcaa tg -# gtgctcaa 4560 - - gtcaaaggta aattgctcac tgtccaaggt aagaaatgtt actttgatgc cc -# acacaggt 4620 - - gagcaagtgg taaaccgctt tgtcgaagct gcacgtggct gctggtatta ct -# ttaactca 4680 - - gctggccaag cagtgactgg acaacaggtc atcaatggta aacaacttta ct -# tcgacggt 4740 - - tcaggtcgtc aagttaaagg acgttatgtt tatgttggtg gtaaacgact ct -# tctgcgat 4800 - - gccaaaactg gtgaattgag acagcgtcgc taattaatat gtactttaaa aa - # t4853 - - - - ( 2 ) information for seq id no : 2 : - - ( i ) sequence characteristics : ( a ) length : 1577 amino - # acids ( b ) type : amino acid ( c ) strandedness : ( d ) topology : not relev - # ant - - ( ii ) molecule type : protein - - ( vi ) original source : ( a ) organism : streptococcu - # s salivarius - - ( xi ) sequence description : seq id no : 2 : - - met glu asn lys val arg phe lys leu his ly - # s val lys lys asn trp 1 5 - # 10 - # 15 - - val thr ile gly val thr thr leu ser met va - # l ala leu ala gly gly 20 - # 25 - # 30 - - ser leu leu ala gln gly lys val glu ala as - # p glu thr ser ala pro 35 - # 40 - # 45 - - asn gly asp gly leu gln gln leu ser glu as - # p gly thr ala ser leu50 - # 55 - # 60 - - val thr thr thr thr val thr glu gln ala se - # r ala gln ala ser val 65 - # 70 - # 75 - # 80 - - ser ala val ala thr ala ser val ser his gl - # u thr ser phe gln ala 85 - # 90 - # 95 - - ala thr ser ala val ser gln glu ala thr al - # a gln ala gln thr ser 100 - # 105 - # 110 - - pro val ala ser gln glu val ala val ser se - # r gln thr gln ser ser 115 - # 120 - # 125 - - gly gln glu thr gln thr thr glu gln val se - # r gln gly gln thr ser130 - # 135 - # 140 - - thr gln val ala gly gln thr ser ala gln se - # r thr pro ser val thr 145 1 - # 50 1 - # 55 1 -# 60 - - glu gln ala arg pro arg val leu thr asn al - # a ala pro ala ileala 165 - # 170 - # 175 - - thr arg ala ala asp ser thr ile arg ile as - # n ala asn arg asn thr 180 - # 185 - # 190 - - asn ile thr ile thr ala ser gly thr thr pr - # o asn val thr ile ile 195 - # 200 - # 205 - - thr gly pro asn thr pro lys pro asn val th - # r val thr ser pro asn210 - # 215 - # 220 - - gly thr arg pro asn val thr ile val thr gl - # n pro asn gln pro asn 225 2 - # 30 2 - # 35 2 -# 40 - - lys pro val gln pro ser gln pro ser gln pr - # o asn lys pro valgln 245 - # 250 - # 255 - - pro asn gln pro ser leu asp tyr lys pro va - # l ala ser asn leu lys 260 - # 265 - # 270 - - thr ile asp gly lys gln tyr tyr val glu as - # n gly val val lys lys 275 - # 280 - # 285 - - asn ala ala ile glu leu asp gly arg leu ty - # r tyr phe asp glu thr290 - # 295 - # 300 - - gly ala met val asp gln ser lys pro leu ty - # r arg ala asp ala ile 305 3 - # 10 3 - # 15 3 -# 20 - - pro asn asn ser ile tyr ala val tyr asn gl - # n ala tyr asp thrser 325 - # 330 - # 335 - - ser lys ser phe glu his leu asp asn phe le - # u thr ala asp ser trp 340 - # 345 - # 350 - - tyr arg pro lys gln ile leu lys asp gly ly - # s asn trp thr ala ser 355 - # 360 - # 365 - - thr glu lys asp tyr arg pro leu leu met th - # r trp trp pro asp lys370 - # 375 - # 380 - - val thr gln val asn tyr leu asn tyr met se - # r gln gln gly phe gly 385 3 - # 90 3 - # 95 4 -# 00 - - asn lys thr tyr thr thr asp met met ser ty - # r asp leu ala alaala 405 - # 410 - # 415 - - ala glu thr val gln arg gly ile glu glu ar - # g ile gly arg glu gly 420 - # 425 - # 430 - - asn thr thr trp leu arg gln leu met ser as - # p phe ile lys thr gln 435 - # 440 - # 445 - - pro gly trp asn ser glu ser glu asp asn le - # u leu val gly lys asp450 - # 455 - # 460 - - his leu gln gly gly ala leu thr phe leu as - # n asn ser ala thr ser 465 4 - # 70 4 - # 75 4 -# 80 - - his ala asn ser asp phe arg leu met asn ar - # g thr pro thr asngln 485 - # 490 - # 495 - - thr gly thr arg lys tyr his ile asp arg se - # r asn gly gly tyr glu 500 - # 505 - # 510 - - leu leu leu ala asn asp ile asp asn ser as - # n pro ala val gln ala 515 - # 520 - # 525 - - glu gln leu asn trp leu his tyr ile met as - # n ile gly ser ile leu530 - # 535 - # 540 - - gly asn asp pro ser ala asn phe asp gly va - # l arg ile asp ala val 545 5 - # 50 5 - # 55 5 -# 60 - - asp asn val asp ala asp leu leu gln ile al - # a ser asp tyr phelys 565 - # 570 - # 575 - - glu lys tyr arg val ala asp asn glu ala as - # n ala ile ala his leu 580 - # 585 - # 590 - - ser ile leu glu ala trp ser tyr asn asp hi - # s gln tyr asn lys asp 595 - # 600 - # 605 - - thr lys gly ala gln leu ser ile asp asn pr - # o leu arg glu thr leu610 - # 615 - # 620 - - leu thr thr phe leu arg lys ser asn tyr ar - # g gly ser leu glu arg 625 6 - # 30 6 - # 35 6 -# 40 - - val ile thr asn ser leu asn asn arg ser se - # r glu gln lys histhr 645 - # 650 - # 655 - - pro arg asp ala asn tyr ile phe val arg al - # a his asp ser glu val 660 - # 665 - # 670 - - gln ala val leu ala asn ile ile ser lys gl - # n ile asn pro lys thr 675 - # 680 - # 685 - - asp gly phe thr phe thr met asp glu leu ly - # s gln ala phe glu ile690 - # 695 - # 700 - - tyr asn ala asp ile ala lys ala asp lys ly - # s tyr thr gln tyr asn 705 7 - # 10 7 - # 15 7 -# 20 - - ile pro ala ala tyr ala thr met leu thr as - # n lys asp ser ilethr 725 - # 730 - # 735 - - arg val tyr tyr gly asp leu phe thr asp as - # p gly gln tyr met ala 740 - # 745 - # 750 - - glu lys ser pro tyr tyr asn ala ile asp al - # a leu leu arg ala arg 755 - # 760 - # 765 - - ile lys tyr val ala gly gly gln asp met ly - # s val thr lys leu asn770 - # 775 - # 780 - - gly tyr glu ile met ser ser val arg tyr gl - # y lys gly ala glu glu 785 7 - # 90 7 - # 95 8 -# 00 - - ala asn gln leu gly thr ala glu thr arg as - # n gln gly met leuval 805 - # 810 - # 815 - - leu thr ala asn arg pro asp met lys leu gl - # y ala asn asp arg leu 820 - # 825 - # 830 - - val val asn met gly ala ala his lys asn gl - # n ala tyr arg pro leu 835 - # 840 - # 845 - - leu leu ser lys ser thr gly leu ala thr ty - # r leu lys asp ser asp850 - # 855 - # 860 - - val pro ala gly leu val arg tyr thr asp as - # n gln gly asn leu thr 865 8 - # 70 8 - # 75 8 -# 80 - - phe thr ala asp asp ile ala gly his ser th - # r val glu val sergly 885 - # 890 - # 895 - - tyr leu ala val trp val pro val gly ala se - # r glu asn gln asp ala 900 - # 905 - # 910 - - arg thr lys ala ser ser thr lys lys gly gl - # u gln val phe glu ser 915 - # 920 - # 925 - - ser ala ala leu asp ser gln val ile tyr gl - # u gly phe ser asn phe930 - # 935 - # 940 - - gln asp phe val lys thr pro ser gln tyr th - # r asn arg val ile ala 945 9 - # 50 9 - # 55 9 -# 60 - - gln asn ala lys leu phe lys glu trp gly il - # e thr ser phe gluphe 965 - # 970 - # 975 - - ala pro gln tyr val ser ser gln asp gly th - # r phe leu asp ser ile 980 - # 985 - # 990 - - ile glu asn gly tyr ala phe glu asp arg ty - # r asp ile ala met ser 995 - # 1000 - # 1005 - - lys asn asn lys tyr gly ser leu lys asp le - # u met asp ala leu arg1010 - # 1015 - # 1020 - - ala leu his ala glu gly ile ser ala ile al - # a asp trp val pro asp 1025 1030 - # 1035 - # 1040 - - gln ile tyr asn leu pro gly lys glu val va - # l thr ala ser arg thr 1045 - # 1050 - # 1055 - - asn ser tyr gly thr pro arg pro asn ala gl - # u ile tyr asn ser leu 1060 - # 1065 - # 1070 - - tyr ala ala lys thr arg thr phe gly asn as - # p phe gln gly lys tyr 1075 - # 1080 - # 1085 - - gly gly ala phe leu asp glu leu lys ala ly - # s tyr pro ala ile phe1090 - # 1095 - # 1100 - - glu arg val gln ile ser asn gly arg lys le - # u thr thr asn glu lys 1105 1110 - # 1115 - # 1120 - - ile thr gln trp ser ala lys tyr phe asn gl - # y ser asn ile gln gly 1125 - # 1130 - # 1135 - - thr gly ala arg tyr val leu gln asp asn al - # a thr asn gln tyr phe 1140 - # 1145 - # 1150 - - ser val lys ala gly gln thr phe leu pro ly - # s gln met thr glu ile 1155 - # 1160 - # 1165 - - thr gly ser gly phe arg arg val gly asp as - # p val gln tyr leu ser1170 - # 1175 - # 1180 - - ile gly gly tyr leu ala lys asn thr phe il - # e gln val gly ala asn 1185 1190 - # 1195 - # 1200 - - gln trp tyr tyr phe asp lys asn gly asn me - # t val thr gly glu gln 1205 - # 1210 - # 1215 - - val ile asp gly lys lys tyr phe phe leu as - # p asn gly leu gln leu 1220 - # 1225 - # 1230 - - arg his val leu arg gln gly ser asp gly hi - # s val tyr tyr tyr asp 1235 - # 1240 - # 1245 - - pro lys gly val gln ala phe asn gly phe ty - # r asp phe ala gly pro1250 - # 1255 - # 1260 - - arg gln asp val arg tyr phe asp gly asn gl - # y gln met tyr arg gly 1265 1270 - # 1275 - # 1280 - - leu his asp met tyr gly thr thr phe tyr ph - # e asp glu lys thr gly 1285 - # 1290 - # 1295 - - ile gln ala lys asp lys phe ile arg phe al - # a asp gly arg thr arg 1300 - # 1305 - # 1310 - - tyr phe ile pro asp thr gly asn leu ala va - # l asn arg phe ala gln 1315 - # 1320 - # 1325 - - asn pro glu asn lys ala trp tyr tyr leu as - # p ser asn gly tyr ala1330 - # 1335 - # 1340 - - val thr gly leu gln thr ile asn gly lys gl - # n tyr tyr phe asp asn 1345 1350 - # 1355 - # 1360 - - glu gly arg gln val lys gly his phe val th - # r ile asn asn gln arg 1365 - # 1370 - # 1375 - - tyr phe leu asp gly asp ser gly glu ile al - # a pro ser arg phe val 1380 - # 1385 - # 1390 - - thr glu asn asn lys trp tyr tyr val asp gl - # y asn gly lys leu val 1395 - # 1400 - # 1405 - - lys gly ala gln val ile asn gly asn his ty - # r tyr phe asn asn asp1410 - # 1415 - # 1420 - - tyr ser gln val lys gly ala trp ala asn gl - # y arg tyr tyr asp gly 1425 1430 - # 1435 - # 1440 - - asp ser gly gln ala val ser asn gln phe il - # e gln ile ala ala asn 1445 - # 1450 - # 1455 - - gln trp ala tyr leu asn gln asp gly his ly - # s val thr gly leu gln 1460 - # 1465 - # 1470 - - asn ile asn asn lys val tyr tyr phe gly se - # r asn gly ala gln val 1475 - # 1480 - # 1485 - - lys gly lys leu leu thr val gln gly lys ly - # s cys tyr phe asp ala1490 - # 1495 - # 1500 - - his thr gly glu gln val val asn arg phe va - # l glu ala ala arg gly 1505 1510 - # 1515 - # 1520 - - cys trp tyr tyr phe asn ser ala gly gln al - # a val thr gly gln gln 1525 - # 1530 - # 1535 - - val ile asn gly lys gln leu tyr phe asp gl - # y ser gly arg gln val 1540 - # 1545 - # 1550 - - lys gly arg tyr val tyr val gly gly lys ar - # g leu phe cys asp ala 1555 - # 1560 - # 1565 - - lys thr gly glu leu arg gln arg arg1570 - # 1575__________________________________________________________________________ 1 . radojevic et al . 1994 aust j agric res 45 , 901 - 12 . 2 . jacques n a , giffard p m , &# 34 ; the glycosyltransferases of oral streptococci &# 34 ; todays life science 1991 ; 3 : 40 - 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