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United States Patent (19) (11 Patent Number: 4,971,908 Kishore Et Al

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United States Patent (19) (11 Patent Number: 4,971,908 Kishore Et Al United States Patent (19) (11 Patent Number: 4,971,908 Kishore et al. 45 Date of Patent: Nov. 20, 1990 54 GLYPHOSATE-TOLERANT 56 References Cited 5-ENOLPYRUVYL-3-PHOSPHOSHKMATE U.S. PATENT DOCUMENTS SYNTHASE 4,769,061 9/1988 Comai ..................................... 71/86 (75. Inventors: Ganesh M. Kishore, Chesterfield; Primary Examiner-Robin Teskin Dilip M. Shah, Creve Coeur, both of Assistant Examiner-S. L. Nolan Mo. Attorney, Agent, or Firm-Dennis R. Hoerner, Jr.; 73 Assignee: Monsanto Company, St. Louis, Mo. Howard C. Stanley; Thomas P. McBride (21) Appl. No.: 179,245 57 ABSTRACT Glyphosate-tolerant 5-enolpyruvyl-3-phosphosikimate 22 Filed: Apr. 22, 1988 (EPSP) synthases, DNA encoding glyhphosate-tolerant EPSP synthases, plant genes encoding the glyphosate Related U.S. Application Data tolerant enzymes, plant transformation vectors contain ing the genes, transformed plant cells and differentiated 63 Continuation-in-part of Ser. No. 54,337, May 26, 1987, transformed plants containing the plant genes are dis abandoned. closed. The glyphosate-tolerant EPSP synthases are 51) Int. Cl. ....................... C12N 15/00; C12N 9/10; prepared by substituting an alanine residue for a glycine CO7H 21/04 residue in a conserved sequence found between posi 52 U.S. C. .............................. 435/172.1; 435/172.3; tions 80 and 120 in the mature wild-type EPSP syn 435/193; 536/27; 935/14 thase. 58) Field of Search............... 435/172.3, 193; 935/14, 935/67, 64 15 Claims, 14 Drawing Sheets U.S. Patent Nov. 20, 1990 Sheet 3 of 14 4,971,908 1. 50 Yeast . .TVYPFK DIPADQQKVV IPPGSKSSN RALITAATGE GQCKIKNLLH Aspergillus . PS. IEVHP GVAHSSNVIC APPGSKSISN RALVLAALGS GCRKNIH Petunia KPS. ...EV, QPIKEISGTV KLPGSKSSN RILLIAALSE GTTVVDNS Tomato KPH. EV, XPIKDISGTV KLPGSKSLSN RILLIAALSE GRTVVDNLLS Arabidopsis KAS. EV, QPIREISGLI KPGSKSLSN RILLIAALSE GTTVVDNLLN Glycine mac KPSTSPEV, EPIKDFSGTI TPGSKSISN RILLIAALSE GTTVVDNLLY Maize . AGAEEIV. QPIKEISGTV KLPGSKSLSN RILLIAALSE GTTVVDNLLN E. coli MES... LTL QPIARVDGTI NLPGSKWSN RAILLAAIAH GKTVLNLLD Salmonella MES... .I.T. QPIARVDGAI NLPGSKSVSN RAILLIAAT.AC GKTALTNLLD Consensus an a we w am no me a m rw me am a munit an rve a PGSK--SN R-L-LAAT-- G- - - - - NLL 51. 100 Yeast SDDTKHMLTA WHEL. KG ATISWEDNGE TVVVEGHGGS TLSACADPLY Aspergillus SDDTEVMLNA IERT.G. A ATFSWEEEGE VVVNGKGG. NLQASSSPLY Petunia SDDIHYMLGA KLGLHVEE DSANQRAVVE GCGGFPVG KESKEEIQLF Tomato SDDIHYMGA KIGHVED DNENQRAIVE GCGGQFPVG. KKSEEEIQLF Arabidopsis SDDINYMDA LKRLGLNVET DSENNRAVWE GCGGIFPAS IDSKSDIELY Glycine max SEDIHYMGA RLGRVED DKTTKQAIVE GCGGLFP.S KESKDENLF Maize SEDVHYMGA RTGSVEA DKAAKRAVVV GCGGKFPW. EDAKEEVQLF E. coli SDDVRHMNA LTALGVSYL SADRTRCEII. GNGG. P LHAEGALELF Salmonella SODVRHMNA SAGINY SADRTRCDIT LRAPGAELF Corisensus SDD- - -M-A - - - - - - - - - - - - - - - - - - - -n - - - - - - - - - - - - - - - - - T 1.01 150 Yeast GNAGTASRF LSAAVNS TSSQKYIVLT APLVDSLRAN Aspergillus GNAGASRF ITVATLANS STVDSSVLT GDLVDALTAN Petunia LGNAGAMRP ILAAVVAGG . NSRYVD SDLVDGLKOL Tomato LGNAGTAMRP AAVVAGG . HSRYVD GDLVDGLKQL Arabidopsis LGNAGTAMRP LTAAVTAAGG . NARYVD GDLVVGLKQL Glycine max IGNAGAMRP LTAAVVAAGG . NASYVD GDIVAGLKQL. Maize GNAGAMRP TAAVTAAGG . NATYVD GDLVVGLKQL E. coli GNAGAMRP LAAA. LCLG S. N. DVL GHLVDALRLG Salmonella GNAGAMRP IAA. ALC LGON.E.IVLT GEPAMLERP GHLVDSLROG Gonsensus LGNAGA-R- - - - - - - - - - as a was emo is aV G- - -M--RPI 151 200 Yeast GTKEYNNE GSLPIKVYTD SVEKGGRIE AATVSSQYVS SIMCAPYAE Aspergillus WILPLNSKGR ASLPLKIAAS GGFAGGNINL AAKVSSQRVS SLIMCAPYAK Petunia GAEVDCFLGT KCPPVRIVSK GGLPGGKVKT, SGSISSQYLT ALLMAAPI.A. Tomato GAEVDCSGT NCPPVRIVSK GGLPGGKWKL SGSISSQYLT ALLMAAPLA. Arabidopsis GADVECTGT NCPPVRVNAN GGLPGGKVKL SGSISSQYLT ALTMSAPL.A. Glycine max GADVDCFG NCPPVRVNGK GGPGGKVK. SGSVSSQYLT ALLMAAPI.A. Maize GADVDCFG DCPPVRVNGI GGPGGKVK, SGSISSQYLS ALLMAAPLA. E. coli GAKITYLEQE NYPPLR. LQ GGETGGNVIDV DGSVSSQFLT ALTMAPLAP Salmonelia GANIDYLEQE NYPPLR. R. GGETGGDTV DGSVSSQFTT A.MTAPI.A. Consensus . P- - - - - - ----GG---- - - --SSQ- - - --LM-AP-A- 2011 250 Yeast EPVTALVGG KPISKLYWOM TKMMEKFG NVETSTTEPY TYYIPKGHYI Aspergillus EPVTLRVLGG KPISQPYIDM TAMMRSFG DVQKSTTEEH TYHIPQGRYV Petunia LGOVEEID KLISVPYVEM TLK LMERFGI SVEHSSSWOR FFVRGGQKYK Tomato LGOVEIEID KLISVPYVEM TLKMERFGV 'VEHSSGWDR FLVKGGQKYK Arabidopsis LGOVEIEIVD KISVPYVEM TLK LMERFGW SVEHSDSWDR FFVKGGQKYK Glycine max LGOVEEIVD KISVPYVEM TKLMERFGW SVEHSGNWDR FLVHGGOKYK Maize LGOVEIEID KSPYVEM TLRMERFGV KAEHSDSWDR FYIKGGOKYK E. coli E. DTVIRIKG DLVSKPYIDI TLNLMKTFGV EIE.NQHYQQ FVVKGGQSYQ Salmonella PKDTIIRVKG ELVSKPYD TNLMKTFG. VEIANHHYQQ FVVKGGQQYH Consensus - - -S--Y--- ---M.--FG tims a w - - as a - Y. FIGURE 2(a) U.S. Patent Nov. 20, 1990 Sheet 4 of 14 4,971,908 251 3OO Yeast NPSEYVIESD ASSAYPLAF AMMTGVTV PNIGFESLQG DARFARDVLK Aspergillus NPAEYVIESD ASCAYPAV AAVTGTTCTV PNIGSASLOG DARFAVEVLR Petunia SPGKAFVEGD ASSASYFLAG AAVTGGTITV EGCGTNSLQG DVKFA.EVLE Totato SPGKAVEGD ASSASYFLAG AAVTGGTVTV EGCGTSSLQG DVKFA.EVLE Arabidopsis SPGNAYVEGD ASSACYFLAG AATGETVTV EGCGTTSLQG DVKFA.EVLE Brassica SPGNAFVEGD ASSASYLAG AATGGITV NGCGTSSLQG DVKFA.EVLE Maize SPKNAYVEGD ASSAYFLAG AAITGGTVTV EGCGTTSLQG DVKFA.EVLE E. coli SPGTYLVEGD ASSASYFAA AAIKGGTVKV TGIGRNSMOG DIRFA. DVLE Salmonella SPGRYLVEGD ASSASYFAA GAIKGGTVKV TGIGRKSMQG DIRFA. DVLE Consensus -P- - - - -E-D AS-A-Y-A- -A--G---V ---G--S-QG D--FA--VL 30 350 Yeast PMGCKITOTA TSTTVSGPPV GTKPKHVD MEPMTDAFLT ACVVAASHD Aspergillus PMGCTVEQTE TSTTWTGPSD GIL.RASKR GYGTNDRCVP RCFRTGSHRP Petunia KMGAEWTWTE NSWTVKGPPR SSSGR. KHLR ADVNMNKMP DVAMAVVA Tomato KMGAEWTWE NSWVKGPPR NSSG. MKHLR AIDVNMNKMP OWAMAVVA Arabidopsis KMGCKVSWTE NSWTVTGPPR DAFG. MRHR AIDVNMNKMP DVAMAVVA Brassica KMGAKVWSE NSWTVSGPPR DFSGR. KVR GIDVNMNKMP OVAMTAVVA Maize MMGAKVWE TSVTVTGPPR SHFGR. KHK AIDVNMNKMP DVAMAVVA DAAMTIATAA E. coli KMGATICW. a us 0 s 4 GDOY ISCR. GEN AIDMDMNHP DAAMTLATA Salmonella MGATITW. e o O GDDF IACTR. GELH AIDMDMNHIP Consensus -MG- - - - - - - - - - - - - G- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 35 400 Yeast SDPNSANTTT IEGIANQRVK ECNRIAMA ELAKFGVKTT ELPDGIQVHG Aspergillus MEKSQTTPPV SSGIANQRVK ECNRIKAMKD ELAKFGVICR EHDDG. Petunia YADGP IRDVASWRVK ETERMIAICT ERKGAVE EGPD. Totato IFADGPT. IRDVASWRVK ETERMAICT ERKLGAVV EGSD. Arabidopsis FADGPT. IRDVASWRVK ETERMAIC ERKLGAVE EGSD. Brassica ANGP. IRDVASWRVK ETERMIAICT ERKLGATVE EGPD. Maize IFADGP. ROWASWRVK ETERMVART ETKLGASVE EGPD. E. coli LFAKGT. LRNIYNWRVK EDRAMA ERKVGAEVE EGHD. Salmonella FAKG. LRNIYNWRVK EDREAMAT ERKVGAEVE EGHD . w Consensus am as a ma up RVK. E--R--A- - - E.-K-G- - - - E--D- - - - - - 401 450 Yeast NSIKDKVP SDSSGPWGVC TYDDHRVAMS FSILLAGMVNS QNERDEVANP Aspergillus LEIDGIDRS NLRQPVGGVP CYDDHRWAFS FSVL. SLVTPQP Petunia YCIITPPEK. N. VDID TYDOHRMAMA FS. AACADVP Tomato YCIITPPEKL, N. VTEID TYDDHRMAMA FS. LAACADVP Arabidopsis YCVITPPKK. VKAEID YDDHRMAMA FS. LAACADVP Brassica YCVITPPEK, N. .WAD TYDDHRMAMA FS. LAACGDVP Maize YCIITPPEKL N. .WAD TYODHRMAMA FS. LAACAEVP E. coli YIRITPPEKE, N. FAEIA TYNDHRMAMC FS. VALSDP Salmonella YIRTPPAKL, . OHADIG TYNDHRMAMC FS. VASDTP Consensus - - - - - - - - - - - - - - - - - - -Y-OHR-A- S- - - - - - - - a m i u or a a P 451. 478 Yeast WRIERHCTG KTWPGWWOV, HSELGA. Aspergillus IILEKECVG KWPGWWDT, RQLFKV. Petunia WTINDPGCTR KTFPNYFDVI, QQYSKI. Tomato VTIKNPGCTR KTFPDYFEVI, QKYSKH . Arabidopsis ITINDSGCTR KTFPDYFQVL ERITKH. Brassica WTIKDP. CTR KTFPDYFEVI, ERKH . Maize VIRDPGCTR KTFPDYFDVI STEVKN . E. coli WTLDPKCTA KTFPDYFEQL. ARISOAA* Salmonella WTILDPKCTA KTFPDYFEQI. ARMSPA Consensus ------C-- K-P----, FIGURE 2(b) U.S. Patent Nov. 20, 1990 Sheet 5 of 14 4,971,908 SYNTHETIC MULTI-LINKER U.S. Patent Nov. 20, 1990 Sheet 6 of 14 4,971,908 CaMV 35S PROMOTER Filed EcoRI 1. o 60 GAATTAATTCCCGATCCTATCTGTCACTTCACAAAAGGACAGTAGAAAAGGAAGGTGGC O A 120 ACTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACA o 18O GTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAA o O 240 CCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAC TATA O . s 3OO AATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGA 5' mRNA d 332 GGACACGCTGAAATCACCAGTCTCTCTCTACA SYNTHETIC MULTI-LINKER BglII ClaI SmaI KpnI Sall. EcoRI AGATCTATCGATTCCCGGGTACCTCGAGAATTCCC NOS 3. 368 e 420 GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGG O - a 48O TCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACAT 3' End mRNA O 54 O GTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACAT s O e 600 TTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGT HindIII TCATCTATGTTACTAGATCggggatcc.gtcgacctgcagocaagctt641 648 FIGURE 4 U.S. Patent Nov. 20, 1990 Sheet 7 of 14 4,971,908 ECOR SYNTHETIC MULTI-LINKER RIGHT BORDER FIGURE 5 U.S. Patent Nov. 20, 1990 Sheet 8 of 14 4,971,908 SYNTHETIC MULTI-LINKER RIGHT BORDER FIGURE 6 U.S. Patent Nov. 20, 1990 Sheet 11 of 14 4971,908 Pst Xma ECORV -des BglII NOS/NPTII/ SmaI NOS SpcR 35S RB T-DNA RIGHT BORDER AAC(3)-IV pMON825 NOS NOS ECORI PSt. RB RK2 Bg|II Klenow Filled ECORI ECORI KenOW Pst multilinker filled pMON 841 HindIII PSt. (35s/AAC3-IV/NOS ECORI ECORI BamHI PSt Xmni changed N Noss to HindIII by NOS 3' Ampf ECO RV dileti mutagenesis /AmpR AAC(3)-IV pMON843 99 VSO-pMON844 pMON849 AAC-3-IV by site directed mutagenesis ECORV BamHI Xmn ECORI KlenOW filled Sindi y StuI (35S/AAC3-IV/NOS) HindIII ECORI HindIII (35S/AAC3-IV/NOS) pMON505 ECORI RK2 Ori HindIII EcoRI pMON845 NOS 3' OS RK2 Ori 7 SSN SpcR 35S HindII pMON851 NOS BCI RK2 Ori FIGURE 9 RB YaNN U.S. Patent Nov. 20, 1990 Sheet 12 of 14 4,971,908 ECOR NOS/NPTII/I N BOII LH A.SpcR s'35Y-9Y Aval 'Spc NOS 3 pMON120 NOS plMON530 BamHI NOri
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