US 2004OO67506A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0067506 A1 Scheres et al. (43) Pub. Date: Apr. 8, 2004
(54) NOVEL ROOT SPECIFIC PROMOTER Publication Classification DRIVING THE EXPRESSION OF ANOVEL LRR RECEPTOR-LIKE KINASE 51) Int.nt. Cl.C.7 ...... C12O 1/68; CO7H 21/04 C07K 14/705; C12P 21/02 (76) Inventors: Ben Scheres, Ac Utrecht (NL); Renze (52) U.S. Cl...... 435/6; 435/69.1; 435/320.1; Heidstra, Lh Utrecht (NL) 435/325; 530/350, 536/23.5 Correspondence Address: (57) ABSTRACT Ann R Pokalsky The present invention relates to the field of plant molecular Dilworth & Barrese biology, more particularly to the root-specific gene expres 333 Earle Ovington Boulevard Sion in plants. The invention provides nucleic acids for a Uniondale, NY 11553 (US) novel transcriptional regulatory root-specific promoter and nucleic acid and protein Sequences coding for a new LRR (21) Appl. No.: 10/433,731 receptor-kinase protein, further Specified as a root clavata 1 homolog (RCH1). Further provided are compositions com (22) PCT Filed: Dec. 4, 2001 prising nucleic acids, polypeptides, antibodies and vectors. The invention further provides for methods for modifying (86) PCT No.: PCT/EPO1/14154 cell fate and/or plant development and/or plant morphology Related U.S. Application Data and/or plant biochemistry and/or plant physiology compris ing the modification of expression in particular cells, tissues (60) Provisional application No. 60/256,204, filed on Dec. or organs of a plant of the novel LRR receptor-like kinase or 15, 2000. comprising the expressing of a gene of interest under the control of the novel transcriptional regulatory root-specific (30) Foreign Application Priority Data promoter. Further are provided compounds interacting with the new polypeptides for use as herbicides or growth regu Dec. 4, 2000 (EP)...... OO87O288.8 lators. Patent Application Publication Apr. 8, 2004 Sheet 1 of 40 US 2004/0067506 A1
Figure 1
Carrant opinion in Part slokogy Patent Application Publication Apr. 8, 2004 Sheet 2 of 40 US 2004/0067506 A1
Primers used in experiments described SECR D No. Primer Sequence (5'-3' Details SEC ID No. 6 LRR1 AAYAAYTTMASYGGTKMDWTHCC Degenerate LRR primer SEC ID No. 7 LRR2 CARYTHAMCGGWGAAAHCC Degenerate LRR primer SEQID No. 8 LRR3 MAYYTSAYGGAHCTATHCC Degenerate LRR primer SEOD No. 9 KIN1 THCCWTTWGSCATGTANTCATA Degenerate kinase primer SECR D No. 10 KN2 AYTCDGGOGCHAHGTADCC Degenerate kinase primer SEQD No. 11 KN3 MAYHCCRAARCTRTAVACATC Degenerate kinase primer SEO D No. 12 RCHF CGATCAGACACAAGAACAT RCH1 specific primer SEQ ID No. 13 RCHR AGCAATGGTGTTGGAAGAA RCH1 specific primer SECD No. 14 UBF TGCAGATCTTYGGAAGAC Compl. to 5' end of repeating ubiquitin unit SEQD No. 15 UBIR GACTCCTTCTGGATGTTG Compl. to 3' end of repeating ubiquitin unit SEQID No. 16 M13F TGTAAAACGACGGCCAGT Compl. to p3S sequence SEQ ID No. 17 PRCH1R CACACAGGATCCAATCGGCATTT Compl. to RCH1 promoter, includes small GCAAAGACATA Coding sequence and BamH site Patent Application Publication Apr. 8, 2004 Sheet 3 of 40 US 2004/0067506 A1 Figure 3
Patent Application Publication Apr. 8, 2004 Sheet 5 of 40 US 2004/0067506 A1
GTCTAGCGGTATTAGGCGTGGGCGGATACGAGCAAACAAATGATTCGAGATGATAACGACAGAGACTGGAGA AAAICTAGGACATGGCAATTCACACCTTTTCAGAAACCAACTTCACAGTCGAACATGTACCAAGTGTTTGGTAGAAG GTAATGITATAGGGAAAGGTTGCTCCGGGATAGGTACAAAGCTGAAAGCCTAACAGAGAAGTCATCGCGGTGAAAAAG
Figure 5-2
CTCTGGCCAGTGACAGACCTAATCGAATGAGAAAACTAAGTCATCAGGAGTTCGAGACTCATTCTCAGCTGAAGAAA AACACTGGACGACAGACACAAGAACATGTACGCTCTTGGGATGTGTGGAACAAGAACACTAGACTTCTTAGT AGATTATATGTCAAATGGGAGTTTGGGAAGTIGCTTCACGAAAGGAGCGGTGTATGTAGCTTAGGATGGGAAGTGAGG TACAAGATTATACTTGGTGCAGCTCAGGGTTTGGCTTACTTGCACCATGACTGTGTTCCTCCCATTGTTCATAGAGACAT CAAGGCAAATAATATTCTGATTGGCCCGATTTTGAACCTTACATTGGAGATTTCGGACTCGCTAAGCTTGTTGAGATG GCGACTITGCTCGTTCTTCCAACACCATTGCTGGTTCCTATGGTTACATAGCTCCAGGTACTTCCCAAATTTCTGTTTTG CTGCTAATTAGTTAACTTGATACACATAAGTACTAGAGCAGCCTAGTAATGGTAGAAATTCAGGTTTAAGT GGTTTGGTTTGGCATTTTGAAAAGAATAGTATTGGTTCAGTGATTTCGATTTGGTTAAAATTTTAGAAAAAA AATGGTAGAAGAAATAATTTTTGATTAATATATACACTACTGIATACGATTTAAAAAAAAAATTGATTGGTTTGGT CTCGATATTTTGCTTGTAGCTTGGTTCAATATTTACTATATATATGATTCATGACTTTCATACTAATCAATATTTGTT CGGTTTCGTTTTGATTAGCTGAAAGTGAATCATATAGAAAAATGACCAACTATCATATACTTACACGCTACAAG ATTTTCGATGTAATTTTGGTGTAAGCATTTIGGTTTTATATGTTCATCCTTAGTAACTAACAACAAATATCAATTGGGA AGCATAACGTTATATATTCCCATAAGGCTATAACACTATAATTATGCAGAATACGGATACTCAATGAAGATAACAGAGAA AAGCGACGTGTACAGCTATGGAGTCGAGTGCTCGAGGACTAACGGGTAAGCAACCAATCGACCAACGATACCAGATG GACTCCACATAGTGGACTGGGTCAAGAAAATCAGAGACATACAAGTAATCGACCAAGGACTACAAGCAAGACCAGAGTCA GAGGTTGAAGAGATGATGCAAACGCTAGGAGTCGCGCTTTTAGCATTAATCCAATACCCGAAGACAGGCCTACAATGAA AGATGTGGCTGCTATGCTAGGAGATATGTCAAGAGAGAGAGGAATCGATGAAAGTTGATGGTTGCTCGGGAAGTGTA ACAATGGAAGAGAACGTGGCAAAGATGATTCGACTTCATCGGTTATGCAACAAACGGCTAAGTATTGAGAAGTAGTAGC ACGAGTTCTCTGCGTCTTCTTTGCTTTACTCTTCTTCTTCTTCTGCTACTTCTAATGTTAGACCAAATCTTAAATAA
Patent Application Publication Apr. 8, 2004 Sheet 7 of 40 US 2004/0067506 A1 Figure 7
Patent Application Publication Apr. 8, 2004 Sheet 10 of 40 US 2004/0067506A1
Figure 10-1 CLV1 1 ...... mamrillkthl. ... lflhlyliffspcfayt. dimevillnlkssm 38 . : . : RCH1 1 MSLQMPIPRKKALTVSHFSITLSLFLAFFISSTSASTNEVSALISWLHSS 50 39 igpkghglhdwihss spdahcsfsgvscdddarvislinv...... 77 : . : 51 NSPPPSVFSGWNPSDSDPCQWPYITCSSPDNKLVTEINVVSVQLALPFPP 100 78 . . . Sftp...... lfgtispeigmilthlvinlitlaannftgelplem 114 . : : . . . : : 101 NISSFTSLOKLVISNTNLTGAISSEIGDCSELIVIDLSSNSLVGEIPSSL 150 115 ksiltslkvlinisnngnlitgtfpgeilkamvdlevliditynnnfngklippem 164 ...... : : . . . : 151 GKLKNLQELCINSNG. LTGKIPPE. LGDCVSLKNLEIFDNYLSENLPLET 1.98 165 selkklkylsfggnf.fsgeipesygdiqsleylglngaglisgkSpafls 213 I. : ...... : 199 GKISTLESIRAGGNSELSGKIPEEIGNCRNLKVLGLAATKISGSLPWSLG 248 214 rlknlremyigyynsytggvprefggltkleildmaSctltgeiptslisn 263 . . . : : : . . : : . . : . : 249 QLSKLQSLFV. YSTMLSGEIPKELGNCSELINTFLYDNDLSGTLPKELGK 297 264 lkhlhtliflhinnlitghippelsgilvs.lksldlisingltgeipos finlg 313 ...... 298 LONLEKMLLWQNNLHGPIPEEIGFMKSLNAIDLSMNYFSGTIPKSFGNLS 347 314 nitlinlfrnnlygqipe ...... aigellipkl 339 - : : : . 348 NLQELMLSSNNITGSIPSILSDCTKLVOFQIDANQISGLIPPEIGLLKEL 397 340 evfevwennftlqlpanlgringnlikldvisonhltglipkdlcrgeklem 389 : : : II. : : . 398 NIFLGWONKLEGNIPDELAGCONLQALDLSQNYLTGSLPAGLFQLRNLTK 447 390 lilsnniff fgpipeelgkcksltkirivknllingtvpaglfnlplvtiie 439 : . ::: : : : 448 LLLISNAISGVIPLETGNCTSLVRLRLVNNRITGEIPKGIGFLONLSFLD 497 440 ltdniffsgelpvtmsg. divldqiylisnnwfsgeippaignfpnligtliflcd 488 I. : . . . : : . . . 498 LSENNLSGPVPLEISNCRQLQMLNLSNNTLQGYLPLSLSSLTKLQVLDVS 547 489 rnrfirginipreifelkhlsrints annitggipdsisrcstlisvdlsrn 538 : . . . . . : . . . 548 SNDLTGKIPDSLGHLISLNRLILSKNSFNGEIPSSLGHCTNLQLLDLSSN 597 539 ringeipkginnvknlg. tilnisgnqltgsiptgignmtslittldlsfnd 587
O 0. d O o h d to e s a e p d 598 NISGTIPEELFDIQDLDIALNLSWNSLDGFIPERISALNRLSVLDISHNM 647
Patent Application Publication Apr. 8, 2004 Sheet 12 of 40 US 2004/0067506A1
Figur 11
AB019227.pro ACOO2334 pro ACOO7651 pro RCH1 pro
TO5050.pro CLV1 pro ER.pro BRI1 pro 110.0 100 80 60 40 20 O Patent Application Publication Apr. 8, 2004 Sheet 13 of 40 US 2004/0067506A1
Figure 12 AC002328.pro APO00603.pro BRI pro AC006532.pro AB007644.pro T48499 pro AB010698.pro AC005967.pro AC002339.pro ABO 19235pro T48489-pro ER.pro TO4587 pro AB016874.pro TO5322.pro ACO34257.pro ACO08017.pro ACO21198.pro AL161513.pro AC005395.pro RCH1 pro AB028621 pro AB011476.pro TO5050 pro ACO 15446.pro AB019227.pro AC002334 pro ACOO7651 pro TO5898.pro T46033pro CLV1 pro TO5335.pro TOO712pro T10659.pro AB016887 pro ABO25613.pro AB025627 pro ACOOO 132.pro APOOO417 pro ACO67754.pro AF1494.13.pro TO5897 pro HAEpro ACO101.55pro RPS5pro RPS2pro RPP8.pro RPM1.pro RPS4pro N.pro RPP5pro 329.6 ------300 250 200 150 OO 5O O Patent Application Publication Apr. 8, 2004 Sheet 14 of 40 US 2004/0067506 A1
Figure 13 SEQID NO 18: 842 bp
AAGCTTAAATGACAATTTAGTACCTTGGGTTGGTCATGATTTAGAGCGGAACAAATATACCAT ACATCAAACGAGGATATACAGAGAAAATTCATGGAAGTATGGAATTTAGAGGACAATTTCTC TTCTGGGCTACAACGGACCGGCCCATTCGCTCATTTACCCAGAGGTATCGAGTTTGTGGACTTT TGATGCCGCTAGAGACTATTGGCATCGGATTGAAAAAAATGTTTACTTCGTTGTTAACAATTTT CTGAATGCAATATTTTCCTTGTCATGAATATTTAAACTTGTTATTACTTTCTTTTAGCTTAGGTG TGGACAATTATGGAGTTTACTTCAAACGAGGAAGAATCTTAAACGCTCGGTTCAGGTCTCGAA AACAAACCAACTCACAATCCTGACTTAATTGAGGAAAACAATGCAAAACCACATGCATGCTT CCATATTTCTATCATAATCTTATAAGAAAAAACACTACTAAGTGAAATGATTCTGTATATATAT AACCAATGCCTTTTGTTTTGTGATATTTTATGTATATATAACTATTGACTTTTGTCATCTATGGA TAGTGTCTCGGGCTCTTGGCAAACATATTTCAAAGAAAAGTTAATGACTGTAATTAATTAATC TGAAGCTAGAAACAGAACCCCGAGGTAAAAGAAAAAGACAGAGCACATGAAGTTTAGTACTT TTATATATTTAATATATCATTCTTTCTTATTGCTTATCTCTAAAGCAAAAACTTCCCTAAACCCT AAGCCAAAGGACTCAGATCGATGCAGAACCAAGAAGGCTTGTTTTGGATTTGAGAGCCAAAT GCAAAGAAAAAAACTCTT Patent Application Publication Apr. 8, 2004 Sheet 15 of 40 US 2004/0067506A1
NCBI Sequence Viewer Figure 14
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1: G '3510337" (GenBank Arabidopsis thaliana genomi... PubMed, Protein, Related Sequences, 1
LOCUS AB017061 61712 bp DNA PN 09-AUG-2000 DEFINITION Arabidopsis thaliana genomic DNA, chromosome 5, TAC clone:K19 E20. ACCESSION AB017061 WERSION ABO1761.. G:35.1337 SOURCEKEYWORDS Arabidopsis thaliana (strain: Columbia) DNA, clone lib: Mitsui TAC clone K9E20. ORGANISM ArabidopsisEukaryota; Wiridiplantae; thaliana Embryophyta; Tracheophyta; Spermatophyta Magnoliophyta eudicotyledons; core eudicots; Rosidae; eurosids II Brassicallies; Brassicaceae; Arabidopsis. REFERENCE 1 (sites) AUTHORS Kaneko, T., Katoh, T., Sato, S., Nakamura, Y., Asamizu, R., Kotani, H., Miyajima, N. and Tabata, S. TITLE Structural analysis of Arabidopsis thaliana chromosome 5. IX. Sequence features of the regions of 1,011,550 bp covered by seventeen P1 and TAC clones JOURNAL DNA Res. 6 (3) 183-195 (1999) MEONE 99397.451 REFERENCE 2 (bases 1 to 61712) AUTHORS Nakamura, Y. TITLE Direct Submission JOURNA Submitted (26-AUG-1998) to the DDBJ/EMBL/GenBank databases. . Yasukazu Nakamura, Kazusa DNA Research Institute, Department of Plant Gene Research; 1532-3, Yana, Kisarazu, Chiba 292-0812, Japan. (E-mail :ynakamuekazusa. or.jp, Tel: 81-438-52-3935, Fax: 81-438-52-3934) COMMENT Address for correspondence: kaos(3)kazusa.or.jp For the latest information on annotation of this clone, please see http://www.kazusa. or.jp/kaos/cgi-bin/agd graph.cgi?c=K19E20 Genes with similarity to proteins in the databases are described in 'product' or 'note’ qualifiers. Genes that have no significant protein similarity are described as "unknown protein' . The software programs used to predict genes include: Grail (Informatics Group, Oak Ridge National Laboratory, http://compbio.ornil.gov/Grail-l. 37), " ; GENSCAN (Chris Burge, MIT, http://CCR-081.mit.edu/GENSCAN.html), NetGene2 (S.M. Hebsgaard, et al., CBS, Technical University of ... Denmark, http://www.cbs.dtu. dk/services/NetGene2/) and SplicePredictor (Volker Brendel, Stanford University, http://gremlin1.zool.iastate. edu/cgi-bin/sp.cgi) . Genes encoding tRNAs are predicted by tRNAscan-SE (Sean Eddy, Washington University School of Medicine St. Louis, http://genome.wustl.edu/eddy/trNAscan-SE/). This sequence may not be the entire insert of this clone. It may be shorter because we remove Overlaps between neighboring submissions. The 5' clone is K24G6 and the 3' clone is K20Jl. FEATURES Location/Qualifiers
Patent Application Publication Apr. 8, 2004 Sheet 21 of 40 US 2004/0067506A1 NCBI Sequence Viewer
1081 ataatgalaga aattatatga to tattotala ataattaatt aggcCaaata ttattotaag 4. tgaaaaatgt cacgctcaat ttctottata taataactCC ttCccttgaa gcctt tottt 1201. aactott to atct tcc titc titcc ttctetc ttct ttaaac a CacactCC gacaaaag CC 1261. totgtctCgg acagaga caa agaala Cagala cagagcaaga acaatggctg ttacacaaat 1321 acttgtcgto tttgcttctg cattacticct atcaatgttc ttcactggCg togattocac 1381 aaggtatCga aatgtttittg agtcaaattit attacgtgat C tggttcatgg alacaaag Cat 1441 tgaagttcto act tctcaaa totaagctaal aacaagaaad coctittittct tgatttgaat 150 tacttgatca tgatta Catg gattttgtat tatagat.cta alatatggagg gagttctttt 1561. tattt tattt ttt tatCata gattatgtag agat.ccatgt gatcaaacac aagttgttgttca 1621 ttagagttta ttgaatgata atgcaa.catt totaatgttgta ttagoagtga ttaa.gcgagg 1581 tttgg tatat tggttttata atttgagttg tgttttcggg tttalactalat gagttggitta 1741 attaatgact tCcaggtocta atgaaaCatg gcatgaacat gCagttgaga. atCcagagga 80 agtagctgcC atggtggaCa tgttgagttitt tottgttgctt aaattttgttg ttata tacta 1861 tatatgaata ataatgattg ttgtttgttc totaattaag tattittagoa totaatgcga. 92. ttggittagtt agtaaaacaa. tttaaaaaat aCactaalact agcattactt gacgttittgg 1981 ...ttggataaat totatgttgatc ttcttittata cattt ttaac tactggagaa aaagaaaata 2041 aaaatcagaa aactggtgaa agttg tattt atgttggagca gggctcaCat ggCaCttgttg 2101 taagaattitt tata cattct gg totaaaagg totaaaaggto aaaatcCata ggg cocaaaca 2151, ccacaccitcg atticcatttc tttcatcactt tttgtttatg tttttgtttt a tattacctt 222 aattictorata atgataaaaa aaatettctict tatgcaaatt ttaactgttt ttalactaaaa. 2281 gtatgttctt ggttcCCgCg cactgactitt ttcaa.cgaga aaatct tttg attatttaga 234. attitt togtt tgoCgtagaa aatgattgaa aactttgttg attgttctat tttatacagt 240 attatgtttc cacatttatc alaatacalaala cgaaalaccta aataggittat aaaagttctga 24.61 ttitt cactta tagaaa.catt CCCatCggag ttatttitttg gttctitccgt. tittct tittitt 2521. gtgttittcaa gattgactac attittgactic cgtaagttat agaact taat gotcaacagac 258 togatttitcg atttgtc.ttc gct tccatgt ttittaattaa. attitt ttcgg Catgaalacca 264 atCalataCat ttalaacaatt tgcaaatCat attgtaaaaC attaaaaaaaa. taaaaatCaa, 2701 ataagtag tg aggataagtt Ctt tatgctt. attct tactg aggggtttca gagittattgg 2751 tggaccagac atgaaa.catg atgcatala Ca tggttcattt agttgagtaa gatttggatg 282 ggattoctitca tttaatgcaa tgcaccCaaa. tCatatagat gaataaattit attitttgtaa 288.1 aaaa.cattalia atattattgc ttgcaaaaat Cttatggagt tattgtagac attggtocala 2941 aalacacgtgt. aatgttcttg ttt ttgttitt tgaagagaaa Catgttgtaaa tgttcCalaca 3001 cCtaatctitt aaaa.ca.gtga attgccatca gCCCCatgca tetc.cctcgt. gtagtgaatt 3061 gttctotgaa tgatttcact cgitatic titta gtoctitatcto gttattactt atalactatac 3121 tag tactitta aagatctgtt tggcacaagt tataatgtta tgactatatg attaaacgaa 31.81 tag tatttitt tggattatta accCallaaa. a catata Caa ctattataac gtocatttctal 3241 agaaagttta gcagtgttitt attitt tattit ttggcttitta gaaatcattg gattcttgat 3301 tgaataggaa tag tttgcgt. aacaalagoaa. gattitttt to aattittct tal Caatgcgtga 3.361 attagaa.gca ttagaataat CCCaCCgacg aalactatttg toaaatgaca CaCattaaaa 342. gtgg tocacc caattcCtect gattitcggitt alatatagtaa ccatgtttat att tag tgat 348 taattctgtc. attaattcaat ttagct tagt gatcatgtta aattaatctg gttctgactaa 3541 accggttttg atggttcagg agcattCgala acagdacago gagaagaaga, ttaggatact 3601 totcatgctic aacCggaaat CCaatcgatg attgttggcg ttgttgaccgg agatggcaat 3661 CCCgg Cgaaa. acatctagotc. aatctg.cgcaa. togggttcgg togcaatgca atcggcgg ta. 3721 gagaC9g tog ttactacgta gtgagtgatc caaacgacga. taaccoggitt alaccCtaaac 3781 Cgggalacatt acgtcacgca gtaattcaag aagaaccatt atggat.cgtC tittaaacgtg 3841 acatggtoat aacattaaaa. gaagaactaa tdatgaacag tttcaaaa.ca atcgatggtc. 3901. gtgg.cgt.cala cgttcacata gCtaatgg ty catgcatcac gattcagttc gtgaCtalaca 396 ttatcattoa tgggatacat atticatgatt gtagaCCaac tgg taacgct atggittagaa 402 gctictoctitc gCattatggit tggagaacta tggctgatgg agatgggatt togattittcg 4.08 ggtcgag tota tatttggatt gatcataatt Cgctt togaa ttgttgctgat gggct tattg 4141 atgctgttat ggcttctact gC tattacta totCcaataa ttactitcaca CaCCaCaatg 420.1 agg taagaat ttgttcticaca ttt tatgttt tttggttgct tgaatcggaa gttagtttitt 426 tt tatgg tag ctctgtttta tgacaggitta tgttattggg acatagtgat acctatacaia 4321 gggatalaggt tatgcaagtt acaatagoett attaatcattt tggtgaaggit Cttattoaga A381 gaatgcc.gag gta Catactt tggcctaact tatgattctt ggcaaaaact togaaaagtC 444 gacaaaataa acatttitt.ca ttaatgcagg tgtaggcatg gatattitt.ca tgtagittaac 450 aatgactata Cacattggga aatg tatgcg atcggtggca gtgcgag toC taccattaac 4561 agccaaggala atagatat ct cgCCCCaaga aa.ccgg tittg ctaaagaggit actittcaaga 4621 gttagg toga Ctagg toaaa taaagaacCg atctt to ttg act tttgact tag to tcaaa. 468 ctataaccot tatact tata gattacagtt totalacaalaat tottacagca aaaact tatg 474 ggatgtttca ggtgacaaag. agaga C tatg Cagg gCaatg gcaatggagg cattggaatt 4801 ggagatcaga aggagatct t ttttaaacg. gagctt tott cacacgttct ggatcagg to 486 ttggagctag Ctacgctaga gct tcaagtt tagcagocaa atcatCatct Cttgttgg.cg Patent Application Publication Apr. 8, 2004 Sheet 22 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
4921 tCattaccta Caatgcc.ggt gctottaatt gcagaggtgg togc.cggtgt tala tag titac 4981 talaagttctat cittcttttgg tttcttgcat tata catggc aatagocacta ttgaatgtcg 5041. aggcttgatt taaaattgcc toaactgtat tataaagttg taatgatttg tttcaccgcc 5101 aattattgat gtatictataa. aagcaaacaa agttggaact CtcCCtttac tctic ticatct 561. taatgtt ttc Ctacacaagg a taaaagaala Catagaaatt tgttcaaaaaa. aaaaaaaata 522 gcagttctdt Cggaaatgca atgtag tatc aataatattg gtgaagttta totaticttggc 5281 atgttgactat ggtttttgga tCagtttggt toggttatat cgcc tatgat tgtttttgtt 5341 cttggittagc tttggittaag agtttcaaaa. cCaaatttala ccgatttitct tatto.cggct 540 alaattggitta tttttggctg aagttggttt tatttbCCgg ttalaattggit totaataaaac 5461 totagtaa.ca gttaaaagaa gtctaaaaat ttggtgaaaa. Cggg totCag tttggtataa. 5521. totggittatt atlala Caaaaa. tacctaagga. cggttcgatC Cgalaccgaac Caalaattittc. 5581 Cgtt tag.cga. tgcaaaatta aagttctt.cc tagaggttcal accgatagaa ctgaaaatct 56.41 tgattcaatc Cggttcggitt ttctcggttg ggittatttat ccattggctg aaatccogca 570 talacggg tog tottattatg taggctaact acaagttggit cattcaaggt taalacctgac 5761 caacCCg tag agtCcgctala CCgggatatg attaagatgg gCtgacCaag actaaccocg SB21 tttggtogag gataa.catcC ttcataatga tggagaggtg tgggg tatgg tgtcaaaggg S881 aataagalagg gatgttgaagg tggtggalacc acgtgag agg agacaagaac CaCatgaga.g. 5941. Catatgagag gaaataaaag. aaaggacctt agitatcattg totatgttabg ttgttgatat 600 tgataagata ggtttggcaa atggcttgct totctttcat tacacact tt gtttcaccala 6061 atgttctott tatgctaatc tcticcittitt tattttctta atttgtcaca tttagcgtgt. 61.21 tttitttittac agtc.gcagag actgcttcta CggCCaCaala attacactat tacggCaaga 6181 aatttalacgc. cgttgagtcg Ctgcgattca gacgacaagt aaaaa.cagtt tttaaagttg 624 acgctgaaat titccagogat tacggaatta ctgtttctitt titc.ttt tect gatcgctgcg 5301 gcaactg.cgg cactittattt titccoaaata Cacatcatect Caggctt titt atttgttgcc.g 536 Cagttct tgc agctg.cgact gttaaacgaa catgctaatt atct talacta aatttaattit 642 gttctottaa.g tgagtaag.cg gagtaattitc ttaaacaatt tgaatag tag gttagataaa 648. attaaaatett aagtttagaa tttgatatto tttitttct tt tgacctgaag tgtacacata 6541 accecgtott aaatagoatc atgalacacat CaacaatcCa tCtcaatttg ttt togtggit 66O1 Ctcgcgaaag acgtacgitac aagtggctaa caattatatt aagcatcctt taalatacatt 661 aagaataata atctitatgtt acagaaatct aagacgtaca gagggaatta tgttacatgc 672 toCggtttag aatatt Cat cacaaaaaat acatttitat ttgtagattit attatatgtta 578. acatttcaaa. agcatgtact ttaatctitt aacaatacat tgcc.gttcttg aaaacaaaag 6841 togaaagaCg totaaatagag aattittgacg toatactact atgttactta ttcCatgaag 690 aaaaCataat atcaaatta gattttgttt tgaaaattitC gaCatagitat tattgtccgt. 696 acttggacgt. attaccalaaaa ccacttgtat taaaaatca taaacaaaaa. aaacticattt 702 atctatgcga. acatalacaca attatcaaata tgttagataa. aaagtag toa acacttittct 708 caccCC titat attg tatgaa ttatgacata atatatagtg tttatagaat tgg tatgaaa 741. ttatttgg to aacaaatata gtotccattt tittgatag to: aaCactatoa. aaatttgtta 72O1 aaaaatcaat agtttttacc Catatag titt acctaccaca ttalaggttitt ttcc to ttgt 726. agtatataag a taalactatt tgacaacaat aaagtaaatt ttacatacala catalagaaat 7321. aatacgataa ttaataagtic ttgcaccatg totacgalaat tattt totta tttcatgctt. 7381 ttgg tagttc tottctCagg taattaactt tittittct tct tottcttata tgctoaacga 744 totaatacgt. caatgtacaa ttagotttga Cattittgaca gaacttcttt a tatttittitt 75O1 Cataggaiago taatgalacta atgctatoat titt tetctic atacatatto attiteattta 7561 aaagaa.gcat actalattitta atgataattic tgtatagttt tta atttaga cacatacta 762 tgatactitta act cactatt tgttgcagta ttaacgatta ttcc taaaac tgagg Cacala 7681 aagagatgta gacaagaact aga.gc.cggga aagcaatgttg tgttggcaaa gtg tagggaa 7741. Ctgttgtt toa alacaattgaa aggatticggg agttgtattg agaa.goCaCC aggCtct tct 7801 aagtata cat gtaattgttct Ctacaattgt ggtoccaccag gattittittta gaacgcaaga 786 taatgtaaac totaatgaaac aatagitttaa, tttitctatgg attacaaaat gaaaattitat 7921 Ctttalaatta Caaataatgt aa tagtttaa. Caaaaaatga aaattitatect ttatct taala 798. attacaaaac ttittatact t gccatattta aggtttgttc ttctgatagt a tattactat 804. attgttgct tit aaccatttitt tgataaacgt. gatgtagcca tott.cct tctt Ctagat.ct tc 81.01 Ctgcatalaca atttaatcac acaaaatcala tittctacct c atcggattat cacagacCCa 8161. tgaacaaatg attacacateca alaccCaalaat tggalag cata agacaa.gcta alagaCtalaga 8221 agattgg tac cctttgcgag atttggga Cg agaaaatago tagaga.cgaa gaatctoCtt 8281 agacgaggag aataga-tacg aggagatago gagaaagttct aaattitcgtt titcccCaaa. 8341 tgttgtaaatt ttaaagatat tttc.cgitatg agctagotca ttatgcCCgC gaacaaaatt 8401 ggtott taga ccatatgatc cgttt tag tt gCgggctgaC Cacaga.ccgt. coacaa tatt 8461 ttggactgag cittagcc.cat ggttggatga ccatttgaca, gttctact ta. ttaaag.cg at 8521 acagottgta Cagctittgac agittacca at CaCaaaaact ttatcaaaaa. cgitta caaaa B581 agttt tactic agagttt ttt ttgtacaact tttittg taca actitt tetcta acagotttgc . 8641 actgttccaa taaagctaac agCtaaaaat CttgtaCaat ttittgg ttac taatcaga Cc 8701 Cattatgtta ggg ttcticcic tact ttgaga. aaaatcagoC caaatgttcta actotgg taa Patent Application Publication Apr. 8, 2004 Sheet 23 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
8761. taaatatttg aatgagttta ttctact.gta tatattttaC acacataaaa. Cttggtttct 8821 tataa atttg aagttttgat attitt tttga ataaaaaCtt tgagaCatala ttoratcattt 88.81. accttt tata ttacaattica tCaagattitt tatt ttatca Catttctaala Ctactgaata 8941 aaagattcaa ttCttataaa. tgttggag teca gtaaagaaac aacagagctt gtgtatacac 9001 agggalaagag tottccgtct ttatgtttag tt toaccalat aatcCataca Cgagcatgag 9061 cCatctgga aatggtgaaa acgtttccgg tdtcgtacCg ttatttittct Cttgtaaact 9121 ttggagatga gtttgatgga Cgaatgacala tocticacaga tacgtaggtt cittaacaatg 91.81. Cg tattggitt tgcCagggct cgtgcttatc aacCCaaacg. ctgtagcgat cittctogctg 924 tgg taatgaa gtacattt to tttatctict tCctcCaagt ttaaCaaaaC Ctgttgttggtg 9301 attgg totat accCagctag totcagtttg totgagattt Ctacgag Cat tgagttgatt 9361 tetttggctt ttgggtgtga atcatect tcc. aCaacaaact Catgaa.gocac gocatcaatg 94.21 togatcaaac tacacccagg gtottt tott atatocttct cCtt Cattct cagoctoatc. 9481 totgaalacct Ctgaccagtt toottgagaa gCatacatgt ttgagagggc gaCataagct 954 ccactgtcat gaggalaccat atCCatCaga a tattggcaa. cgc.gcttgcC Catctogaca 96O1 tttcc.ctgca ttctgcaa.gc aCCCaaaagt gctttccata toacatcatc tggittttata 9661 ggca tattaa gtataaactic CtcagoCtca tdaagaagaC CCgaacgacC Calataaatca 972 accatacacc Cgtag togttc. aatcCtaggc totaalaccat CCaCactCaC Catctgactg 9781 aaatatC toC ttcottctitc. tacCaaCCCg cCatggCtac aagcagtcaa. aagattaatg 984.1 taggcaa.cat Cactagg tog aactcCagcc tgcctoattt tacaaaaa.ca gtocaatagoa 990. teacctgcct ggc.catgitat agcaaacCCg ttaatcatcg cacticcalagt aatcacgttc 996.1 totctaggta acctic toaaa. tacatgaatc gctt totcaa tgatcCCaCa tt togaatac 10 021 attatcaatca aag CagagcC aagcacatCa totaatcctaa ttccactato citctgcatac 10081 aaatgcaa.cc. attctoccaa citctaalagac cCaagc.cgtg aaatagocagg aagaaCacta 0.141 aCCaaagtala Cataattcgg totaatatocC cct ttcttical totcacgaaa aacCtcaa.ca O20 gcatct ttga aaaa.ccc.gtt cagogaataa cc.cgatatoca tog tattocca Cgaaacaacg. 10261 Cttctittgac goattittatc alaacaa catc. cgtgcagott tacaatcatCC aagttcticata 1032 talaccatcaa. totatalacatt Cocacaaaact atttcaccat citctott to t. totatoragtic O381 at CaCCaCCa tatic tetctic tataatgttc tataaaa.ca acacacgagc atct ttcata 10441 aaaCCaCaCa tdacatacat totalacaaga ttacticatca CalaattcatC aCCaCCalaat O501 ccatact tca aa.gc.caaacC atgaatctgo ttaccttct gaatct tcCC agttittocgca 1956 caa.gc.cttaa galacagaggg aaacgtaaac Cgattcggitt CaacaaactC acacticatc 10621 atctogtaga ataatgttgat tgcgatcaga gotttgttctt cgtcactotc actgaaacct 1068. Ctgattatag tattocCaaga gaa.gcaatitt cgttgaggota tttgattaaa. gatcttatgt 10741. gogtag toaa. grgtcacggtg gtggagatcg gagg tagcao agaatcggag aatctotgcg 10801 gCggcgagag tgttctogcat ttgacCagat ttaatgaaaa CggCatggat. ttgagagagg O861 tctottatag titcgacagtt gttgatttga ggaaag agtg aagatggatg actogo.cgga 10921 galaga.cgc.cg gCgagttacC goCgggaga.g aaaagttgttct gtgttgggitt Cattgagaat 10981. Calacaaagat toctacgtgt ggctatgtala gacgCataac aagatataat alaagattgttg 1041 gtcgaaaacc gattaalaccg. alaccgaataa. accgattitat totagttatcg gogCtgaagc 11101. tgCaaggagg agttaa.ccga. togg.cgaaat tattittggaa tttcggtott tggatcgg tt 11161. cggttcggca actictgaacC gttttgttgt Catatotaala gatagittaga actictalaccg. 11221 aataa.ccgaa taatct tatg catttgcgca gacaaagaga. tttgattatt tatgaga.cala 11281 gaggalagoca agttgggact aalactaatgc aaagcttago atgagag toa cgcaa.gcaga 1341 agcttagaag cacattccac aaatcc.gacc titta Cactac tactat tact actacalaata 11401 ggCttcaa.ca ttagagactal tgcactitcac Ctttgcaa.ca accatttalag taCatacata 11461. citccoaaaat ggctgcct ct gtggaatact titccCtatta citcacCaCCC toccatcaac 11521 atcctic ticcic ttcticcagtt ccaccoccitc. ct toccacat titc.cccticca ccaccticcat 11581 tetctoctoc tecatcaccCa coaccaccac attittetctics toctocatcag CCaCCgc.cat 11641 CaccatatcC tCatcCtcat CCgCCaCCaC CatCaccata tocticatcCC catcagotcac 1170 CgCC toocaCC teacgttctt cCacca CCaC caccalaccacc agca CCgggg catcatgtta 11761 toattgttggit tgttatctot Cttggaagtt tgttttittct tgcattcCtC getgetgcto 1182 to bitctgcta totcaagaala agaagaaagt Cttctacaaa ggctgagatt atcgagtttg 1881 atgalacacct CaaagttcCaa gaalacca tag tgcaaggacc cCatggcgaa Caaacga gag 1941 tgg taatgct tgaag aggac attcatttgg tagaggatat totacaag act gaaaagctta 1200 gcc.gtcc tag tCaccticagt toa actggaa gaCatgcCat tgatatt tea gatcCCaatc 12061 atcattt tac tgagcaaaag to Ctaaatca aaaggctCaa aatgtaaa.ca atcatattta 121.21 ttcacaccat Ctalactaata aaaacaatitt aaatatgg tal Ccaaatcttg CaatcCaaaa. 1218, cittgatattg attoccaagat aaatgctgta Catgtc.cg to caattaatac aacttaatat 1224l tt tactatga ttbccactitt gcgaatgtaa gta acctitat a tatt tatga tcattgattic 1230 cgtttgctgg Cttttaatgt agatt CC tag tgtactctgt -ttggtttctic aataa Catta 2361 ttccatttala Ctcgagttcto agatgtt tot attaaataag taatgaaag.c agctictaatt 12421. CtcCocaaaat toa caagtat agaaagatag aaagatagaa acagagittat tgcatggaag . 12481 Ctgaaaatca tacagcatgg tggatataac Caatgaga.ca Caaaacaatc acggCatgttg 12541 ttatagalaca a Cact tat ca. totg tattaa cataaagcaa. aagttcc tat acgaCatagt Patent Application Publication Apr. 8, 2004 Sheet 24 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
1.2601 CCaaagttac agacCatalag CaCaCC titat gatttgttgt aCCC totalaa aca Caggatg 2661 atgacaagaa gCalaatttgc cCalaatttac tgagggaaaa. acgagggctt. alacgctCcca 12721 tatCtcaaac aaaaact tct caaacagttt Catgttgttca gattggaggg CaatggCaac 1278 ggataagctg ccatcattag taggacttgg tagcacaaaa gacaaacCCt cgitatggaat 12841 toCaCCaggit CCCataaaga taggacgaCC cCaaCCaaag totgcatcat. aaatagg tala 12901 tCtalaccCag cttgttgattc cCalaatttgg gCacttgtag g tatgtgcac CgCggaCaag 12961 ggctgacaga taggctgca totCcaggta gtCaagagct gacctcagat agttatcatc 13021 CatacgaacC aagaaatCat gaatctgtcC tgcagoatac Catgttggct tagataacaa 1308. atCCCCtgca acagozcaatg gtgttgcagt gaatatocaca ttCccalaagt alacCaggagg 13141 cagotgcgga Cgtag to tag acctitcCatc agttgcaatg tacagtttcg totcttggtc. 1320.1 gtttggaagc cotcgc.gc.ct ttcc.cactga totCcacaca tgccctgcca aCatctogta 13261 tgag Citg tag ctgacagtgt to CcatcCtc Cttggattitc gCCttalaga.g Caacaa.gctg 13321 gtctCgtgtt aatttgaata tagagaCagt gg tattotoca gg tectgatt taga.cggatc 1338. aag aggtatC ttca tacttg gtgcaggctg attattocaa.ca tgatgaaaag Caggctgttgg 3441 Cgg gttcCcta gCtcggagga. gtgttcgatc aatgaaaggt ggaatggitta ggteaaga CC 1.3501 acgagcCata tdagaCCatg tgttgataaa atgaagaCCa galgaalaccat CtgcCgCgtg 1356.1 atgttgcatc cCalla CCCCat gtgaagctOC ccCacattta aagaaagttca CCtatatoat 13621 caaacaatat cattacataa ctaaaaa.cag alacatacatec atcaaagaat atcattacag 13681 aactaaaaaa Cagaacaa.ca Caagcattca ataaattaaa. alaagggaaag. actittaalatt 374. aagctgacct goaaaacgag aag.cgggaala gag tgaatgc cagoggagtg atcaacttcg 13801 ggaataagct gacggagatt aaggg tagga gCaaaatcatC caaaatcatc gata acagaa 1386. ggagtatCag CCaCaacgaa galgalacacca gcaccgttac aatcgatctd. aatacgacca 392 to atcgtctic tottcaagcg accagoCata ggg taaaaag gaCaagggc tttggaaaga 13981 gct tcc titca tgacctgagg gtcaaagaala ttggaagcgc cggtggg tot gtagaagtag 14041 acactagggg tatggaatct ggggatgaca aggtogacgt. tggagttcCa aagattagtg 14101 attggtgttct Cggtggcagg CCggaccatg gtggaatcto tgatgttaat ttitcatattt 1416.1 ttaagaaaga toaaacgaac aalaccalaattit tgttttgagt ttgatctgag aaattgttgttg 14221 agaa.gogagg agggg tag at ataagtgggit gtgaacaaaa. aaggaaaggt. cgtaatcCtt 1428. Caccitaccgc. ccoacgaCCC cgcc tactcc. atctgtcgac caacticcic agaaggttta 14341 toattggtoc atctoraticac toggtoccitac ttt totatoc ggggttggta aa.cccgttcc 14401 citctgtcCta aggcCCalaat aatactaatg ggcttaaata gtataaagtt cgaatgaaga 1446 agalaggalaaa. aalaattgaCa agttgattgt tatagctacce gtacaagtta gtCaatcgaa 1452. tgccaaagtt tggCttaact talacticatta ccacgtagoa gag cataa.ca acalaccacca 1458 atalacatcta acggCgtaala ctoacgtgtt alaga gaatta cgt.ca.gaatc caagaaagttc 14641. tttittt toag tacaccacala a taaattatg ttgaattitat atc.cgaacta tactatatta 14.701 tatgaaaa.ca taccticaatt agtCatalaat ttcacatttic atttittaatett attalactaat 14761 ggttgttgtt gCggatagot tttct ttgct ttctgttcta alaattatttic ttagoCatta 1482 tttgatgatc Caacttggitt tgaggat.ctal ataaaaattic aatttctittg aa.gcattcat. 14881 tggcttaatg atccaaacct tttgtcCtcC cactctobtaia gaaaataaag Caaaagcaaa. 14941 actaaaagaa aa.gcaactag taCCgtgggt tgg toaatgg talacccatgC taccalaatac 1500 Caatcaaag.c ttaaatgaca atttagtacC ttgggttggt. Catgatt tag agcggaacaa 505 a tataccata catcaaacga. ggatatacag agaaaattoca tggalagtatg gaatttagag 1512 gacaatttct Cttctgggct acaacgg acc ggCCCatticg, cCattacc cagaggitatc 15181. gagtttgttgg acttittgatg cc.gctagaga. ctattggcat Cggattgaaa. aaaatgttta 15241. Cttcgttgtt aacaattitt tgaatgcaat atttitcc ttg toatgaatat ttaaacttgt 1530. tattacttitc ttttagctta ggtgttggaca attatggagt tact tcaaa. Cgaggaagaa 1536 tottaaacgc. toggttcagg totcgaaaac aalaccalactic acaatcCtga. Cttaattgag 15421 gaaaacaatg Caaaac CaCa tgcatgctitc Catatttcta tdataatctt ataagaaaaa 15481 acactactaa gtgaaatgat totgtatata tatalaccalat gcc titttgtt ttgttgatatt 1554 titatgtatat atalactattg actitttgtca tCtatggata gtgttctoggg Ctcttggcaa. 15 601 acatatttca aagaaaagtt aatgactgta attaattaat Ctgaagctag aalacagaaCC 15551 CCgagg taaa. agaaaaagaC agagcacatg aagtttagta cittttatata tittaata tat 15721 cattctetct tattgctitat c totaaag.ca aaaact tcc.c taalaccctaa gCCaaaggaC 15781 tCagat.cgat gCagalaccala galaggcttgtc. tittggatttg agagcCaaat gCaaagaaaa. 1584 aaactictta t gtc.tttgcaa. atgcc.gatto Calaga aaaaa. agcct taact gttcticatt 15901. tittcc attac attgttctotg tttittagctt tottcatcto citcgacctct gcatcaiacca 595. atgaagttctc. agct ttgatt to ttggct tc acag Ctcgaa ctcgcc.gc.ca cCgtcag tot 16021 to tccggctg gaatcCttct gattotgaCC catgtcaatg gccttacatt acttgttcct 1608. ccticagacaa caaactcgtt acagagatca atgtcgtctic tgttcagtta gctttacctt 1641 to Cct cotaa catttct tcg tt tacticac ttcagaaact cgttatctoc alacactaatc 16201 to accggagic tatatoctetct gagat.cggag attgttcgga gcttatag tit atcgatttaa. 526 gcticaaatag tottgttggit gaaatacctt cgag to tagg gaagctcaag aatct tcaag' 16321 aact ttgttt aaacticcalat ggcctCacag gaaagat.ccc accggaactic ggCg actg.cg 638 ttagcCCaa gaatcttgag attt togata actaCttatc cgagaatcto CCgttggagc Patent Application Publication Apr. 8, 2004 Sheet 25 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
16441 toggaaagat citcgactictt gaga.gcataa gag.ccggagg aaactoragag CtttOaggga 165O1 agatcCCgga ggagatcgga aactgtagga. atctoa aggt cittagggitta gCagocaa.cga. 16561 aaatato.cgg ttct ttacct gtttcgttgg gtCaactaag CaagctcCag totttatctg 16621. tCitat to tac aatgctictoa gg tagatcC ctaaagagct tggaaactgc totgaactta 16681 toaatctgtt totatacgac aatgacttgt CCggCacgct tCCaaaagaa ctaggaaag.c 16741. ttcaaaacct agagaagatg cittctatggc agaataatct CCaC9gaCCt atcCCtgagg 16801 agattggatt Catgaaaag.c ttaaacgc.ca ttgatctotc tatgalactat ttCtcaggaa 15851 CCatCCCtaa atcgttcggt aacttgtcga atctocaaga, got tatgctt agcagoaa.ca 1692 aCatCaccgg gtocaatacct togattictaa gtaaCtgcac aaagcttgtt Cagttccaga 1698 ttgacgc.caa. totagattitca ggitttgattic Caccagagat tggattgctt aaggagctica 17041 acatc tetct gggatggcag aataagctag aagggaatat toCggacgag ttagotcggitt 17101 gtcagaatct tCaagctCtt gacttgtcac agaattatct Caccggat.ca ttacctgcag 1716.1 gtttgtttca gctt.cg taat ctgactaage tottgctitat atctaatgcc at Ctocggtg 17221 ttcatCccact ggagat.cggg aactgcactt cgCttgtcag atta aggctt gtCaataa.ca 728 gaatcaccgg agaaatCCCt aaagg tattg gatttct tca gaacct tag.c ttcc togact 17341 tgtctgagaa CaatctotCC ggtocagttc citttggagat aagta attgt agaCaactCC 7401 agatgcttala Cttgagcaac alacactCttc. aaggttatct toctotgtct ttatcatcgt. 17461 taacaaagct totalag tactt gatgtctott cCaatgacitt gactggcaag attacctgata 1752 gtctaggtoa tectCatttca citcaa.ccggc totattoctaag taagaattct ttcaatggag 1758 alaatccotte gtotecticggit Cactgcacga. atcttcagot tottgatcto agoagtaata 1764 a Catctotgg aactataccg. gaggagct tt ttgaCatCca agatctagac attgcgittaa. 1770. acttgagctg gaatt Catta gatggcttta tcc.cggaaag gatttocgcg cittaa.ccgat 1776 tatc.cgtgct cgatattitcg catalacatgc tttcaggcga. Cett totgcg Ctgtc.cggtC 17821 tagaaaactt ggitttctotg aa.catctoctic ataa.ca.gatt Ctcaggittat CttcCagata 1788 gtaaagttgtt tagacagotg ataggagcag agatggaagg aaacaatgga Ctctgttcca 17941. aaggtttocag gtCttgctitt gtaagtaa.ca gttcacagtt aactaca Cag Cgtggtgttgc BOO act.ca Catag acticaggata gCCattggat tgctaatcag cgtgacagog gttctagogg 8061 tattaggcgt. gttggCggitt attacgagcta aacaaatgat to gagatgat aacgattcag 18121 agaCtggaga. aaatctatgg acatggcaat toacacctitt totagaaactC aactitcaicag 88. tCgalacatgt acticaagtgt ttgg tagaag gtaatgttat agggaaaggit tgctCCggga 1824 tagttacaa agctgaaatg CCtaacagag aagttcatcgc. ggtgaaaaag Ctctggccag 1830. tgacagtacC taatctgaat gagaaaacta agtCatcagg agttcgagac to attctoag 1836 Ctgaagtaaa. alacaCttgga tCgatCagaC acaagaaCat tgtacgcttic ttgggatgtt 18421 gttggaacaa galacactaga cttctitatgt atgattatat gtocaaatggg agttctgggaa 18481 gtttgcttca Cgaaaggag C ggtgtatgta gottaggatg ggaagttgagg tacaagatta 1854 tact tggtgc agctOagggit ttggcttact tgCaCCatgal Ctgttgttcct cccattgttc 8601 atagagaCat Caaggcaaat aatattoctga ttggCCCtga ttttgaacct taCattggag 18661 atttcggact cgctaagctt gttgatgatg gogactittgc togttct tcc aacaccattg 1872 citggttccta tggitta cata gctCCaggital Cttcc.caaat ttctgttittg ctgctaatta 18781 gttalacttga taCatCataa gttactag ta. gtcttagoct agtaatgg ta. galaatticagg B84 ttta tag titt ggittittggtt tittggcattt tgaaaagaat agtattittgg ttcagttgat 1890 tttcgatttg gtttaaaatt ttagaaaaaa. aatgg tagaa gaaataattit ttgattaata 1896 ttatatcatC tact.gtatac gatt taaaaa aaaaattgat ttggtttggit ctcgatattt 1902 tgCttgtag C ttggttcaat attt ttacta tatatatgat totatgactitt catactaatc. 19081 aatatttgtt cggitt togtt ttgattag to tgaaagttgaa toatatagitt aaaatattga 1914.1 ccaatctato attataettac acgctacaag atttitcgatg taattittggit gtaagcattt 1920 tggitttittat atgttcatcc ttagtalacta acaiacalalata toa attggga agcatalacgt. 1926 tatatattoc Cataaggcta talacactata attatgcaga attacggattac tcaatgaaga 1932. talacagagaa aag.cgacgtg tacago tatg gag togtag t gctcgagg ta ctaacgggta 1938 agcaa.cCaat CgatcCaacg. attaccagatg gactCcacat agtggaCtgg gtcaagaaaa 19441 totagaga Cat acaagtaatc. gacCaaggac tacaa.gcaa.g accagag tota gagg ttgaag 1950 agatgatgca aacgctagga gtc.gc.gct tt tatgcattaa toCalatacCC galagacaggc 1956 ctacaatgaa agatgttggct gCtatgctta gtgagatatg toaa.gagaga gaggaatcga. 1952. tgaaagttga tggttgct.cg ggaagttgta acaatggaag agaacgtggc aaagatgatt 1968 cgactitcatC ggittatgcaa. caaacggcta agtatttgag aag agtagc acgagtttct 19741 ctg.cgtottc tttgct ttac tottcttctt cittctgctac ttctaatgtt agaccalaatc 1980 ttaaataaat gCatggg taa ttatgttt tt aagttgaacaa. gttt Ctatgt Ctaaagttitt 1986 tgttittatct atatatgacg ttgagatgaa Ctcgg togttt CaaaCCaaga aaaattalacg 1992 gagactaatc tog tottacc titt tacattlet tottccgtota aggcgtcaag ttgattittbit 99.81. tttgaaaatc aaataaagaa aaaactag tig aaalacagata tgaggagg to atalactCata 2004 actgttgactic acgagataaa. tcatgitat.cg tggctgacgg toacggalacc ttttgc tittg 2001 tacccattitt ttcgaggttt gtcCaggaag gcggata att agtg tacctt Cactatatac 2016.1 totata actt aataattitta tat caat tag ata Calaatgt. tta attaaaa aataaaatag 2022. Calaa CCCtta acCaagacca gtgttgtata t taagtaacga aataaaattg aacagalaatt Patent Application Publication Apr. 8, 2004 Sheet 26 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
2028. gttgaCCaga taaataataa gtatagattc atgttt tag t t t t t t tittaa. cittcaattitt 2034 cagattitt tt ttgtag tota aagtttggitt gatgaaaata aaataaaata aaagaaaaat 2040 Ctatatttac ggaalatatat ctCttacaaa. taattctttg caaatgtgga agacctatta 20461 tacatataga catata tatt alacacalateCa. ttatgagtaa, gtaticttaat attataa att 2052 gatgaaaatg ttaaaaaggit tataaatchta tata tatttgtctatttalaa alatggttgaa 20581 aatctitect tt tdaaaaaatc aataattgtt tttatgaaac agtgaagctt aatctttaag 2.064 aagtCCCtca aacaattitt aCCataaaat aagattitcaa attataa Cat acctaatagt 20701 tttatctata tattotalact atCcatgttc ctatggtata a taatatott Ctalacattat 20 761 ttittgaaact tgtttctotc tag tttitt tt ttitt totcaa agcgagatcc aagtttattt 20821 Caagag ttac Calacatalaac aatggcaa.ca aataaattitt tatC tatttit gttgctitt Cg 208B1 ttgatggcgt ttgctgctat totgctgcca atgattitCag g talacacatc tattatctat 20941 taaaatttgg tttgtaatgt tatgataaat aattCtactt taaatatgaa agttaccgtg 21.001 tgtttctatg taalaacaaaa Cottalacctaa ttittttgttg aaggittittaa. ttagctCatg 21.061 ggtgttaaac atalaactato aaaaaatata acCaaaaaaa taaagttttt aagttgtata 21121 tttacttitta tgttgaaaata titctacticc actacgacaa aatgtcactt ttg tagg ttt 21181 tttgttgaagg ticacaaattt tagattacga. gtgcaattag agtalacacaa a tatttgatt 21241 ttgaactaaa. cagtcgtggt tttaaaaata gtgttcaaaa totagaagaag aaaattgcac 21301 tatgtag taa gaaaatagtC gttctattgaa attgatcaat ttt tattitta gag togttta 2.351 atttgtttgt. ttataaactal aacagga Cala atCataa.cat gtttaccggg cgaatgtaca 21421 aacCCatCgg aatgcaatgc agcttgcaaa totaaatggat acaaaggagg ggCttgttgta 21481 agcatgagca taggottcaac aaccggtgct tgttgctgta aacctaattt CaaatCtcaa 21,541. gattoctitt.ca aatctaatga tatcattatt aataataatt agtacatcat. tagatatatg 21.60. tattittgata tgaataaaaa. tatattocaaa tatgtttoga tattgalacca totataatat 21661 ggatalatcat. tCgtaagtta tag tacgatc caaacatttg atcgtatata ttaataaag.c 2721 a tacgaatgc tgaaattitat gttalactata citcatagaaa tittatttata totgccatac 21781 caatacalatt taaaagattt ttcaattaala tgagagataa ataatttatt ttgattattt 2184, ttttgtttct gttttgttcaa Calatatgtaal tatgc.cgatt Ctaaaaaaaa. aatcatactic 21901 tatatcatat attgttctaac cctc tagtta gttcaaacaa catalagtaag ataacaagaa 2.951 ttaaaaataa atttaaatga ttittgatato attatattgaa ttaaaattitc. aaattictato 22O2 aaaatticatg attaaattaa. CacatcCalala tgc.cgaaaaa aaaaaaaaaa. aaaattaa.ca 22081 CatcCaaaaa. ttgtcCacaaa togtactaala ggccCagtaa gtc.tttttgt. CaataagttcC 22141 attaa.gocca acaaacctict tottctottet cgtttitatga catcaatcca tttgaa.gcaa. 22201. acalaat Caga Cgtaaccgaa Caaaaaaaaa. aagttggittt gatcgatcgt. taalaacCtaa 22.261 gaaatggatc. CaaaatcgCC gga atttatc attgatcago cgcttaaaat Cttaggattit 2232 g tattogatg agctotcc.gc caccagagtic to:cggccacc tocaccotaac tgaaaaatgt. 223B1 tgcCag CCgt totaaagtCtt gCacggCggc gtatcggctc tyat.cgctga ggCactc.gcc. 22441 agtCtcggcg CCggaatcgc. ctictggitttt aaacgcgtog Ctggaatcca tototctatt 225O1 caccaccitcc gtc.ctgctgc totcggcgaa attgttct tcg ccgaatctitt to cagtCtca 22551 gtCggcaaaa a tattoraggit alacctacaca taaCactgac caatacaCat atatacgcag 22621 CtatCtttag actittaatgt atataaaatt a tattalacta gaaaagtttt tag taatatt 2258 aaattittaat attgttgttcaa agtaatgtaa. aaataaattt tttittaaatg taaaaaaaaa 227A aattaatatt ttgttgaaga agttcaaatat aattotgcaa tttgagagat alacatalagat 228O1 alaattittcac gtattagtaa gtaaaagttga Caagttatgt -tttatalacca ggtgatttitt 2285 aaaaaatgaa atttgttttg atgcaatato gtgttgaaaag taagaaaact alatatatgca 2292 attaccatatt taalattgttga acct tattaa. tttcaccitaa aatgaaaatt ggaaaactitt 22.981 ttcatgitatt tgatagacitt gtgagtttga attgtataaa titt.ccggcaa. gCaaaag Cag 23041 Ctcgtg tacc aagttgtcgg aalactittaat tttgttagca togcattggac talaacCaacg. 230 caaa tattitt attttattta togtag actg aaga cctaala gagtaaaatg aatgttgaaag. 2361 tgaCacgttg tgttggagtat tttgttatcta actaggtgttg ggaggtoccgg titatggaaag 2322 ctaagaaaac cgagacaccg. gaCaacaaaa. taatgg tatC aa catcacgg gttactic tot 23281 totgcggittt acctataccg. gaccatgtta aagatgctcc agacgagctg aagaaagtta 2334 tatocalaatt abaaattcaa attgtaticta totcattttgttaggaataa gaacaattitt 234.01. agtttggittt tattg tacga. accaaacgtt ttaataaatt gttgtaccala acatgagata 23461 ttatalagg tt ataaaaattg aCaagttatt gtggg tt tag tttgtag atc at tttgaaat 23.52. atag tattala tagacitattt tgttgaaacg. tCcaaatcgt. atagatgggg taacaaaact 23.58 alaacticcitt a tatgattct tttgcataaa. attctgtc.tt tg taataact gaatcataca 23 64 tacttittta gtttgatacg catalatatala ttaccacaac tatgtag togc atttalagata 2370 tect ttect tcca tattatalaat gtg tatcaat accatttaala tott.cgtata Calaagaaaaa 23761. aaacaagatg gatat tag tit titt.cgatgga gatattattt ggttgagttc tttct tcttg 2.3821 aga Calacaac atcCagtgttg gag totaaat gttcCacaat accatccatt tgtataccc.g 23881 tggCttalag C aaatctgatc acatacgctt gtacacticgg accgata aca CabiggcgtCa 2394 tgaacgtCaa tagcgttctat tttagctgta aaagata toca taaaaatc ta. ttatatatga 2400 tagatcatca tt tatgttta tatttagaga. gctcta Catt gagatt.cgat Cacgatcaaa 2405 Cgg talagg tt actitcacgat aalactalacga. aga tag tigta t tittgctgaa tgatg talaat Patent Application Publication Apr. 8, 2004 Sheet 27 of 40 US 2004/0067506A1
NCBI Sequence Viewer
241.21 tatattaaaa aaaaatataa. gg tatatata taaacaaacg. aagg taatat ggttctagatc 24181 aagaaaatac Cctaatagaa atcaaaaagt ttgttgtatat gttittatgaa tttgtcaaatt 24241 gaCttaccgg cCatc.gcaaa. caagagaaca attgttatgg cagtocagtat atgttgaatgt 24,301 ttctggttct gcattttittt gttittgaggit gtcactagot aatagotatt actttgtttt 2436 citctttgttt ttagatgitat cCattgagat taaagtgctc ttatataggg gttctggttitt 2442 cgCagataaa attaatcaat ttagctggat tttattoctat aCCCaCaaaa talaatttata 2448 aaagctaaga tata attttg atatgatggt toatgaaagt gtocaactittg gttcagattta 24541 aacctgcata acgcattatg aaattgacta tatatatatt aagaaagaga. taattcticat 24601 ttaatttitta gtaaaataat tactCaatitt tatattata C Cagalaacaaa. gtgggttcaa 24661 aaattattaa. ggctatatoa tatgtaaata Caattaagaa acatttatgt aagaalaatta 24721 gaaactgaaa. tt toatatoca agaattaa.ca aagaaaa Cat taataaaatt tottgttacc 24,781 ttaaatatac cattt tattt aaatgaacat taaatcatag tattt tagat ttalagaaaaa 2484.1 agalactacac agaaatttct ttaataaatc taatatatat gaaaataaat agaaagCact 2490. actgaaagaa gatcCatata aattaaagat agttctggtota atgCCaatca toattittgaa 249s CCalaatccac atttalaggaa aattgatgtt atticittaac CCgaalaa Cat gttcttgttct 25021 ttgcacatgt gtCtcgc.cct attttt tatt tottcattca acaagttcaaa atgaatctat 25 OB tt tactaCat gatctotata aacgaatgtc. ataaagattit tatggcct tc aalacttgg to 251.41. toaa Catcga. totatgtatat gattatctga ttcatCcata tagaagaggg Ctaaccogcc. 252.01 cCttg tacct tgctittggcc ...ttaaaaaggit tttcaagaat t tactittctt tect tatttga 25261 ttttalactitt gtaaatgtca catttittcc.g ttaattaatt citttcgctga gtgacaaata 25.321 tgttaatatg acatttacca totalagatata tatttitatica tatttttgat acatgactat 25.38 caaacaaacg. toacactatt ggtocagagat tattogacat aatccataag aagttgtaala 25441. tatatocatca agtgtgaata aatgtatata aa tattatta aaaaaaaaaa. ttagtagttg 2550 citttittcgc.g attcgtgcga atagattoctg atgccaaatt tggatattat talaga.gocaca 25561. tgtttctoct tgttgtcgtgt cittlettattta agagagaatc atcatctic tic actictoratict 25 621 totagaagtt aaaaCatCaC gccatctt.cc. gaattaalac gCCgatgttc. tgtcgttt to 2558. tgtc.tcacat attggaacga. gcgtaaagat gcctaattitc totagttaacg. titcccdaact 2574. citcatctott tacagtacaa aaacgc.cCaa agtgagaatg aatc. tatgttg cCgatcaggit 25801 gttctgataaa aagcttctgt ggagagatat gtCaacgaag atgaaattitC cittctittitt 258 61 tgctg.cggaa ttacctgatt tgaggaaaag taacaa.gagg aggggatcto ttaggatgat 25921 Caagttgcaga gcc.gc.cggag ctgacggtgg acgcgtggct gttggggatg atgtgtttitc. 2598 ggittactact tottctaagt atgaagttga citatctgggit caaagtacta agggagattt 26041 gaatctoaag cttgaccotc ttcagtcatt tggtatatat acaatct tcc. tttttgttitt 2601 gttagttcttic gaaattcaga ttggttcttag tgaaatgttgg gatalaagttt tggtoctittgg 26.61 ttagggaact tgattittggit tgatgtttgt ttaattgaga taaataagag Ctaaaaagat 26221 cgaacctttg ttgttgaatgrt cacgttgtat gagcaaaatg talactittggit gtatctatgt 2628 aggagatggg caggctacat tggagggtoc Cattgaggag gtago.gagaa Cagaggctoa 263.41. agcggctgaa aatttgatta gagagttggg tatCCaagtt cgittatctaa toctitacaac 2540. tttgttgttat gtatttgaag tttgagtatic CCtttgttgc gaaaa.catga atg tattgag 2646. ttatttgttgt Ctgg'tttitat atggaCttitt ct tttitctitt tgttgtcacat totcatgcct 26521 gg tattgcag ggccCtttct Ctgcaicagoa ctictoctogg ggtatattitt gtag tog tac 2658 attgaatctt cggtCCatta gtgcaattgg atatgatatg gattacactt tgatgcacta 2664 Caatgtcatg gtgagttct t cct ttcticag acataagtag galaa Cattta tgcacccata 26701 tittgatgcta attitatgg to ttgttcaggc ttgggaagga. aaggott tatg actattgcat 26761 ggaaaatcta aagagcatgg gtttccctgt. tgatggactt gettittgatc Cggaactgg t 26821 gatatgttta ttttcttgttg aaagttgaaa. ggttcCacat tgtag tacco tatCtag titt 2688 ttagagtact caaactatgt tittctict tat tataggittat Caggggtcto atgattgaca 2594 aa.gagaaagg taatttagtt aaggcCgata gatttgggta tgttgaag aga goCatgcacg 27001 gtacaaagat gttatcaaat aaagctgtca ggtatatttg act ttetcaac aCCattgg ta 27O6 atgttgaagaa aaaagagagt tgcaaagttca ttgttctgtt ttgaaggaag totcattcto 2712 ttattgttctt tg tatggatg aaagtgagat Ctatggalagg gag ttagttg acctg.cggaa 27181. ccagag toga tgggagtttc totaatacatt ttt ttcagtt totagaggcto tggcttatgc 2724 aCagg tacgt. tottt tatta Caagaaatgg tagctgaaca atttgcagtt gattittggac 273 01. ataatttact ttatttattt atttgtaact Catataattit totacatt.cgt. tttcticaact 27.361 gatgactcitt caccocaatg aact ttct tt titta caaaat tettcCgcacg gCttctgcta 27421 agcaatgaaa gttcaaatca tttcagatgg ttgatagatt ggatgatgga tittattitcgg 2748. cag atct tyg cacticttgat tataaaggac tgtatalaggt tgttgtt totat agaacCtaaa. 2754 tttcattgaa Ctgaaatct t tgcatatata tttitttaatc taatcatttit ttcCaggctg 276O1 ttgcaaaag.c totct tcaga gca catgttg aagg acaact talaggttgat agtttgtc.ca 27661 tticcataatc attt tottaa. atatocagttt to tccggtoc Ctcttgaata atagt tact t 27721 tgcagagtga gataatgtcc aa.gc.cggalac tatttgtcga. gccagacCCa galactaCCtt 2778. tagctictttt agatcaaaag. gagg taatgg ttcagtct tt acacalatata totalactatg 2784 atggitt toag ttgttaata t tgtcatgttc gttcataggc tgg taagaag Ctcttgct ta. 279 0. toacaaactC ggattatcac tacacagaCa adatgatgaa gcattcattt aacaaattcC
Patent Application Publication Apr. 8, 2004 Sheet 29 of 40 US 2004/0067506A1
NCBI Sequence Viewer
3.1801 tttctoratga agcaa.gacgg tetta tactca atgatttcta tgctgctgct tatgcgacac 31861 aacctgatgc a tattaccgt. to Cttggaaa attataaaaag catttcCCct gatgct tata 31921 Cttggg ttat Cgaaagtgag cCtcticcatt gggcaaatgc tttgtttgaa ggaga.gagat 31.98 atalaccacat gaactcCatt tttgggctag atttctacag ctgggtttct galagcacatg 32041 agttgccCat aactCaaatg ataga.cgaac ttcgagcaaa gttgatgcaa. tcaatatata 32101 CCCatCaggit acaatcCCgg gaatggattg tttcaacgct alacaCCaaCC alacgaagaga. 321-61 agctacagaa agaaatagaa Cttgcaaggt Cact tcaggt CtcagogCCt Cacalatagot 32221 tattocgaggt tCatggtgaa accatcaact tagttgacat caatcagtgt gattgcgact 3228 gtaagg tatg gagaCtaacg. ggtttaccgt. gtagcCacgc. agttgcagtg gttgaatgca 32341 tagggaaaag CCCttatgaa tactgctCca gatact ttaC Ctcagagagt taccgtttaa. 32401. cgitatgctga gtocattaac cctgtc.ccta acacaacaat gacgatgatg attttggagg 324 61 agc.cg CCCgt tgaagggg.tt gtttcggitta CtccaccgcC CaCaaga Cita acaccaccag 32521. ggaggcCaaa. gag talagoag gtggaaccgc. tggacatgtt Caag.cgtcag CtcCagtgta 32581 gCaactgcaa. gggtttagga CaCaacaaga agacctgcaa. agctgttatct talagtattag 32641. ggaga.gaga.g. agaaatagag agga.gaga.ca tggittaggac aaatttgtag ttatatagot 32701 gactttgcc.c alacattttgg ttittggttct totgtttgtt gg gtttittga gaatgat Ctt 32761 alaaggg taaa. aatctoctitC aggttgaaac ccaaacgtga agaga.gcaaa gCggctCaga 3282. atctotgttc Ctaaatgg Ca ttaaacttga tgaaagattit taCataaatt tttttgtatg 3288 taatttgatt tttalacaalaa aatttcaatg aatctttit.ca attacatata ttttgttattt 329 41 Cgagctttac aaaatcCaCC acaCttgtta aaaagctitat gcagttgcc.g acaa.cactitt 33001. gacgtttaca tCcaaaaat. gatcgatgtt catatgagcC tataataaat gCtagaga.ca 3305 gtaagatata agtatcaaaa gagtaaaatc to taccalaga aattgacticc alaccalatcgg 33121 agg taalactc. a taggccaca. alaggtta Cag Caccagoagg agcagott to acagtogatg 3.31.8 gCaCaatgcC tttgtacagal ccatgccacc cttctgatat Cataatctgt Ctalaga.ccgt. 33241 Ctalacatgtt totgitatgca cgtc.gctcca CtcgggctoC attatcttgga tgtc..tctgta 333 01 alacct tcaat ctgcaacaala agaaact ttc. aaaatctato tggcatttct attatttcat. 333 61 gtttgaaggc aattgcattt teattgegtt ggagatcCaC tggcaagttct gaaaa.gottt 334-21 tatatttalag aaatgttt Ca Cacaaattgg agttttctitt Ctatagg tac ggtegacagaa 3348 Caagtggggt. accocggaatc agtaagtaga Ctcagoaa.gc acatatorata gggcagtaaa. 33.54 tgttgaaagtic tag totalaga tacatalagga tagaccCaaa aCatgaaaCC a Catgaatac 33601. gataagttcat tgcgctagtt Cagtg tagga gCattgtaat taCCctagot ggtttgtagt 33661 aga tagtaga tgttgttgaCa acticagocaac tatagttgag taagttagca agaga.gctaa 33.72 gtggctaaaa. ggaagtttgg gatgttgtaat taggtogagt agagattctic attaatcaag 3378. tc to tctatg atgttgtcagtt Cttgctgaag tgaataaata aggagtttaa tagtttggat. 3384.1 cctatoacat tatct tcc.ca gagaatcCat tgggtaaaga ttaactgacc tggaatcgtt 33901 ttttgaccac at Caagggga tggcagacga. gCttggcgct agtgc.ccgc.c cCaag CCCaC 3396 aaatgaaaag Ctggaagcta gaaaggttgg tgtccacatt gatcgggatt ttggaggata 34,021 atttgttatcg atticcagtcC tgtttaaaaa taatcaattg agttgattat Catatgaaca 34081 ttgatcacac atalaactata tattocctatg citcttaccat catcCaacgc etcaaacaat 34141 Catatgttgcc aaattgcagg cCag.cgtaag gcacaatcto aacaag agtt ggtgttaatc 342.01 Cattata caa. tootcittata cctc.gagact gaataatato gaCaaaagttca gaCCtcattg 342 51 ttggatacac ctgttgacag toatcag toa gtcatctata tgtcact taa gCatgaataa. 34.321 ggaagaaaaa Caagaaaatc cccagtacat ggaatttaaa ttatattaca ggttcatalag 343 81 toacgcaact aacCttaggc toccottgttg aggccalatat tgtcCCttaaa. agatcaaaag 34441 gatalagatcc tagagtag Ca gCaCatCctg CtaacgctCc actgacgaag gatagataag 345 O1. ggCtcaaatg tatatgatcc totgcattgc Catalagatca Calaccagota atalagtaagg 345 61 alaccacattg atctotalaat tatacgagtc. aatticcaaaa. gctattoalala gaaatacaca 34621 attaacaaac agaalatgata cCCctaagat agccaccaga CtaCaacaaa aaatagoact 34,681 ggcaatctac caaattaccat atctagagag accgcaatgt tactCagtag actalaattac 3.4741 Ctgttittagt agaaccagat gCaaaagatt toag Cittatg tag tactgta aactgaattg 34,801. aagtata Cgg Catga CCatg agcaaagctg gCaCattacc cc.gc.caaaac CCCtalaagaa 34,861 aacaagtatC aaa.catcaaa acagagaa.ca agacctcata aaaactaatg Ccacaaacct 34921 gaalactacta acatactata aacaaaaaag aaatggitttc agaaaacgga aatgttcaag 34,981 agacggaccc. gala aacct tc ttctor taaat attatctt tag togcctgtac Catticcagta 350 41. tact tagaag CtcCtgaCaa gtttc.cccga aCCaaaCCCC atgaagttcgt. Cggttctago 35101 tgaacctgca at Cacatata acatt Cttca accatctota aatatataaa agctaacCat 351,61 tttctaacaal tgataattict Catctaaaac citcCalcalata CaatcCaaag. Cascaiacala C 352.21 aaactcaaat attott tagc taalaccalaat aaaaatctaa actittatcac toagaggata 35281 gaaaaaaaaa. gccaaacCtg aaatctaatc ttaatgacat Caagaggaga. agttacagaa 353 41 Cg agaaacac Caccagagat agctccggca gaagcatcaa toaga.gcacg tittaatctgt 354 Ol CCC.ggctCat catcaa.ccgt. CgCCg togta ggaatcata t ttt to taggg ttt ttgg to t 35461 Ctataagcta aaabcaatta aaaaagtt to caaaa.cccta tcatcaatat aataaacCC 3.5521 aaacaaaata aggaaagaaa. Ctalaattcaa ggcaacaaat aaaatcaaaa acgaaaactg 35.58. aaatct ttitt taccagataa. agcgagaaac gaaaaggg to gacattgatg gagitt toCgg
Patent Application Publication Apr. 8, 2004 Sheet 34 Of 40 US 2004/0067506 A1
NCBI Sequence Viewer
51001 ttgatttgga gttgttittca aatctgcaat agggaCCtag a taattcata actggagttg 51.061 actitcaatitt attgagttta Ctgaaacaaa. aatgaatcgg agalacgtgca aattgttgcaa. 51121. tggaCaaagg aactgtaata gtatagagca aattittaggc taagctgttc acagotgcaa. 51181 CtgCaggcto aggtggatta gCaacaatga. Cactgaagola gog tocacag agag taggat. 51241 ggtoagaaaa tgatCcgaCC tggCCCgagt aattccagoa CCtCtcacac ttaga.ccc.ct 5,301. cggctcgtga CaCCCCaatc. cacactttgt tectCtccCtc CaCatactict cctgttgttgtt 51.361 gCacactgct tatCatctoc ttctocattg aaga caataC ttcaacctgt aatatgcatc 51421 Cacatcagtt toatggctat agtCtca tag aattacactc agcatgatca aatctosaact 51481 totaatcgaala attgttggata ataattgagg aatttgagat gaatcaccat CaCCaagtat 5154 aactaaaaac a Calatatatt gtaatticcitt Catacctatt tCittagggat. ttalaagggct 51601 tttcgagagt gactitctata gatctgatca gtacatgttgt Ctactgctac aaagtactict 566. tottttaa.ca tctaalacatt tagcatttct Cttggggtoc aaatactata totaggataa 51721 agaaCCagac tggattaaaa. CalaattcCgg tttgttgcCta atcCtattoca cactaaatct 5781. tt tacttatc. tgttittggga agagcc tact tg tagttctgg CCtgaactitt acacagaaaa. 51841. accacggttc acataaaaac ggccCtact t gttgatggtc accatgatto taatttatct 51901. taagctataa Caggaatatg aattt to Cag atalaggagat ttagtgctta Cttgttgatgt 51961 tattgaatatg cgttgcagag tgtcagocto gttctttgct tcactCatct ctagtaattit 52021 tgcago catg CCtgcatctg Cag tatgcaa. attacacctitt gCttctaaac tggalaccaat 52O81 cattt toetcal ttacgtgcta gctcCagcac tttgttcacc tCagtcctica actgtcacac 52141 aalacaccata agtatt tota agatcc tocat taCaaaaagc goatcaagaa taaggoCagt 522O1 aaaacaaaga ataaacgtaa aagttct tcc agagaaattit Cgataatgcc. attatcataag 52251 aactgattac acgatcaact tgacataa.ca togagttgaa tattoctgata ttgttgtaaga S232 titt Cattgag ttcatctoca Ctaagtagct aaggaactCt at tact ttaa. ttacaag Cag 52381 agtagagagt atacaatggg Ctgaattgct gagttttitcc gaattgattt aggagag titt 5244 tgatacctog agaa.gococtitt gCCagaagag aa.catct tcal gCaggalaatg aaag.ccattg 5250. ttcatcaaa gtaggcCatt taagttctgaa gaCaaattct gCtgcagagc CatCttcatt 5256 totgtactica aatggaagat totgccagaC atctt.ccgct aggtgaggitta cgattggagc aatcacticto alatatggata aaagatgttgtc. tgaaagaact gtttgacago ttettctggit 52621. gaaaaaagat ttittggtoca gattacgttg aatagocacag 52681 aaaacttgaa gtacccCtga agatgagaala 5274.1 acgtaittaag aa.caggtota cataggttct agCaaatatc aacCaaaaga 528O1 Cttacccagt ataCagtcta totttagota tatcgaagta gaaatttgac agatcaacaa 5286, ttgttgaatcg ttgtatgatc tgaaataaag aaagctCatt tag talacaca gatgatcCag taagcCtCat gaggttcaaa. gg to cattgc aaatacatca accticactict 52921 aaCatgaaat ttaaagctitt S298. Caccittaatg gatagtgatc. Caattgctga actaaagttt cittagatata 53O4. taactgataa attaacticct gtaagagtga tCtatgtctg titt CCCgtoca caggaaaatc acCaaaataa. gatggagagg ttacctggaa tattittaaag 530 aatacataCg tgaatgatgtc. Ctgaaagaga 53151 aactgg tagt totcgtaiaca ttcttgtata ttctt talcala cattctgaag 5322 gCatgctgat Calatgatagg taaatcCtgg tatggalacag cattatcaac Ctacaaacca 5328. acacaagtta aatattaa.ca gattgtcacc aaCCCCagala tataaaa.ca.g aa.gcaagaag 5334.1 totttga Caag tgggaaaaga ggtgaaaaac actactictat gtaaag Citta agagittagala 5340. alacc tactict cCaatcatga agattitcc.ca agagglta tott taacgttccc cittaact tcc 53461 tatatatatic agacatttga Cgaagaatct gggg accitat tag taCatcc. CCtgttgtaat cgcatalacat Cagctocgta agcaggtgcg toctgttgta 53.521 Cta Cactaga aaccCaaaga taCaatggga 53581 agaaga Calala cccatgtcag gaala Cagatt aaataacgaa gatagttcta S364 tttacaatgt gacaaacctt tgaattitttc cctoctetcala tgaCCaaacg. tggg toCact 53701 acgttaccta gag acttgct cattlettcata ccotecticate CCaatacaaa. to catgttgtt 5.375 ataa.cagoag aatagggggc tttitcctata tacaatcatg Caaaatgttga agaaaaaaat 53821 CaaaagcCaC acaaagatag ctaaaaatct gtagaataat Ctcaattitat aaggcaatat 5388 catggcatgc acttcticacc ttgcgttgcg atgcttgtta acaaagaact ttggalaccat 5394 Ccacgatgct gatctgtacC ttctagataC acgtotgcag ggaaactaag acct tcgcgt. 54OOL tooctaata CaCCagcc.ca agaagatcCt ggaCatacag atgggaalaga aagttgatgta 54.06 aaa tagg to t tgggtgctta gtgttgacitat Ctgattittaal ttagcttctt ttatcatagt 541.21 acctgag toa alaccagacgt. ccatcgitatic agtc.ccct tt toataatcag ctgctittatc tdaggaagta tgacatatac CaCCaC9Cat cactaccott 54181 acga tatttic gg tottccac cacago Cagt S424 ttgggaaatt a tagctgc.ca gaCaagg tat totaaataagg tta Cagaaaa. 543 O. ataataacaac Caataalaa Ca caccatactic tottataggc Coctetacatt aa.catggttg 54.361 attgttctott cattcatgag tggttct ttt gtcttalacat gataaaaag.c aggaataggg 5442 acaCCCCatg toctittgtct tgagatgcac Caatcagatc. gactggaagt CatggCagat 5448 at totgttta citgcc tyaat gaCaattgcc aaaagataac aagtaccalaa attaaactcc 54S41. aaacaatata tatgcatcto ttaaaaaaga taaagtalagg aaaga Cgcta agt caag Cag 545O1 tacgg tatca tecticalacatt gaaaaaatct cacagcaagg tggat.ca.gat catgttggttc 54.66. atgtgctatg aatc.cct tt Cacagotaac accoacaCat gatact titta gCCCaCatala 54.72 tgaagaaaaa. aaaatcaact tataaactica g tatg tatto alactagg tac Ctgatgttggit 54781 accoattitta cattgttgat tgcatccatt gtggctgttgc gaaa CCC titc aacCgatgca Patent Application Publication Apr. 8, 2004 Sheet 35 of 40 US 2004/0067506 A1
NCBI Sequence Viewer
5484.1 aaccactgot Cagtc.gctot aalatatog to ggtttcttag ttcgc.caatc atatggg tat 54901 ttatgagctg Catgacaaga aggalaattaa. atCatgagac tctataggaa atgtttocto 54,961. aaatggttcc toagaaagca tttgaatcaa actCaCCata agattct tcc atgaccagag 55O2. aca tatttitc. atcCagg tala ctgacaa.ca.g cgg tattocCC ttctoCalagg acagaga.goco 55081 CtC taalactg to cagottct tcagtgaact ttCCttcatc atcCaCCgga gagaCaagag 554. goagc.ccgta Cttcaggc.ca gttgcataat CctCctgacc atgacCaggg gCagtgttgga 552.01 cca accotgt to Cagattct gtagttatgt aatCaCCCCC tatalacg act ggacagtc. to 55261 tgttgttcaat gggatgagtg tacctacaac Catgatgaca Caccataaac taaggggttc 55321 aatttatago attacatgca aalactggaat ttcttittatt Ctectaactta aaatgtalacc 55381 tgcagttt to aagatctgaa CCaaggaatg ttt tactitat gctaagct to acaccCoat S5441 togct tctaa tgctggcaca agg togg tog CtaCaatcac gaaaagctitt tgttgattict 5550 ttagaacctt CCCgggaatt ttcttitt tat tgcttgtcac tgttgattica tottctgaga. 55561 atgactgcac titcCacaa.ca gag tactgaa gctttgcatt CaCCgCg aca gCtgaaagtt. 55621 agacaagaaa gaatagaaga agag toacta aatgttgCtta tatagoatt.c aaaaaagg ta. 55681 cgitat tattg Cttctaagtg gagaa.gagat atgagaaaga acggataCata agtaaagtag 55741 gatgcaattit gaCagctgag agaaacacat attactaatat aacCCCttac CagocattggC S580 tggca tag to Caaggtgtag ttgtccatac tgccalagtat a tattaggga taalacticatc. 5586 taaaaggctt. gttittcgctic CCC Ca tittaaaaata gCatatatgc ttittggaaat 55921 atgaccctCc ggatactaca Cataatggca gataCatgttg ggactgatat Cagatalacaa 55981 ggtaaagtgt ggaattgtca aaatattatt totatgaaagc ggcatgcgtt gagaa.ca.gca 5604 atagaaaact acattalaatc agggttcatt ggtgataCat aaagaattag tottcgacta 56101 atggacaa.ca gatcgcatca tggtaaaatg totaacggaag taagaaagag aataggalaga 561.6 Ctgg tatgac tttagoagac ttaagttttg cattgaactg gcc.gtaatat tattattatt 5622. Cttagattat tgattocaaat ttacctic tag ttcagottet goea agag cag toc.gagatga S628 agggCtCCag tgaactggitt tcctacctict ataaatgtaC CCtttgcaagg cCatctggcc 56341 aaatact tca atctacaata tatgataaaa. atatatgttg actittatcag gotttgaaga 5640 actictaataa toatctocta cacggittatt toatccagta aataatgttgg gacgtgagaC S646 Cattacctga gctgcttcat. attcCggatc aagagittaga taaggattgt tocagtctgc 5652 coca tactc.ca aatcgctgta alacaCCalaga ataatgtcat. atgttgctaca tgttcaatcga. 56581 attaccaaacg. aaatatotitt Cttaattatt atgtc.ctgag agatatalaga atcattatga 5664 cggttgcatt caaala Calaga aatCagatat gaaaacaaag cittgatagoc acct taaatg 5670 attcCatttg tgttttgact gttgettittg Caaattitc.gc. cgcctttgcc. Cttaattitta 5676 atggtgtcaag titcct titctt. actitcCtgat CCagggactg Cagaactgaa acgttctgacg 5682 gttacaccala attacacagg Caagtaagta actggaaaat aacaaaag Ca cagttact tt 56881 ctgtt totaa tgacitcaaag. gtccatalacc tattgttgttgt ttacct tetcal actCalattgg 5694 aaggcCatgg caatcccacc Caggaacgta ctgaact tta tag titttgga gCagctgcat 5700 galagalagoag gCagtttgat gagaaggaac tocactattg gacgtaagga aaataaagaa 57061 totCctaagga gagtaaatgg atgtcCaagt aggaaagaCa aatacct tat agogattaat 57.2 gatatcCttC agtatct tgt talaga.gcatg acccatatgc agatcacCat tggCatalagg 571.81 aggaCCatca tgaagaatga aacticccctg aaattictaac talactatoag acacCag tot 57241. Ctcaaagcaa. agttggCata agataattitt galgaaactica citccattgtt attatcagaa 57.30 actictottga agacctgg tt ctic titcc.cac aacttctgga gttcaggttc CCttgttcaaa 57.361 gaatticgctic tCataccalaa aCCagtCttt ggcaaatcaa cc.gtttgctt gtact titcCa 5742. tottcttgct titt.cccctaa CCaCaCaCe acggtaaatg agtgcCacat tgcaata tag 57481 tattaagaaa. galagtacaag Caatgttgatg caaacCttca gatgctttct tggCtgcCat 5754 alacaggcCCa Cgaga CCttc gttittgatga atgacCaaac totattgtttg gttcagttga 5760 aaacCgtgag aCatataaga atgaaaaagc Cttgacggaa gacct tccct tggcaagaaa 57661 agaatcCaac ggCgtgtt to totetalaatt tgaacaactt ttacctgaca aalacCtaaac 57721 ggggaCaaac tacatecticaa. aalactatt to Cacgcaaatt ttcagota aga Cacaataact 5778. totatoeagat tgata taggit gg tagaagta, gtt totccaa. acaagttcet gaga Caacac 57841 atttittctaal gaaagaaaat agag cataga ala CagaCtta gtttggittala taagggctitt 57901 aag cattaca gaggacaCat atgagctcgt. Catgttgatgc tCagatag to agaactCaag 5796. taalactactt gCaattitcat tcttcaaaga tatalagoa at ggataaaagt Calaccalagaa 58 021 gCCtctgaala ttittatcgaa caactggtgt gcaatag taa accittaagaa Cacaaacaat 58O81. aacagaaag C gaac tactitt aacgaalaccC acaaaccgaa attgccalatg cattgataat 58141 alagalaccalat tog togacaa. atgttgttcta ggaatcgaac ttgattagaa agacitatcac 582 01 titt CCCCagc aCaCaaaa.ca aagag toaac agagcagaga. tggattcaaa gtaatcaaat 58.261 ttacattcac atactctg. ta. agatgaagat tgaaccatcg ccattgcago agct tcc.ctg 58.321 ggattitccc.g cgaaggattit gaagaaagac gaCatctgct tcaagtt tot Ctctgtc.gct 58381 totatictato agagctcatt tt cacgg.cga aacaaagagt gagaaggaag gaagaaga Cit 5844. aacgtCacca aatticagg ga. ag.cg tatgttg tg tatgttgttg tggataactg aaaagttgaa 5850. actalaalacca aacct cagaa citcggitt toa atttittggat. tttatc.cgtt aaatcc.ctaa. 58561. atctottgtt tittt tott.c aagct catala tagtaaaaga taatcacgtt taaataCata 5862 aata tactaa Caagtala Cala gagg.cgaaat tctt tta aca tg tatgg taa atagattaala Patent Application Publication Apr. 8, 2004 Sheet 36 Of 40 US 2004/0067506 A1
NCBI Sequence Viewer
58681 tataaacaaa. Caataa.ca acCattagat aaggcgggtg aattta tagg catatatocat 5874. cagtaatgca agatataa.ca gttagactical acagtagcatc. Caccagtctt Catatogaac 58.801 aacttggtoc gtgcttcgac atalaccitcta gcaaaccgga acattccact gccticCalacc 5886. accggcattt CCCtalaccitt ggacatcacc gtgttcc.gac caagaatcgt tatgg tactc 5892. CCgttg tact toCCagtCtt aaaag.ccaag ttcatcgc.ca toaacaa CCC tacticcitt 58.981 tgggctgcte Caacgta Cat CCCttgggct tgacccacca Cagtcgagtt aatgggcaca 59041 totaatgtta aagcgttgtC tatCatggtg attgatcCga aatatgagga acttgagtalt 5910 tttgcaa.ccg. gccCttggat CCtgatggaa cittgggitttc gaccagttac aatgtcgtgc 5961 CaatagaCaC ggagatgttgt. gag tittctot tttttgcc.ga ggCCgaggtg ttitt.cggittt 59221 attgttcttg cgaagttct tc accgttittitt CCggctaagg cggttgoggt gaggaggagg 59281 atttgttgcgg tgaggatgag aatgagctitt gaCatgttag ttttctggtg tog tacttta 59341 attgttittat ttgttgttga tagaatgata gtttatgtaa attgttgggtg tatatactgc 59A01. aactgtgcaa. cgtgctatat atcatcgagta atagatgaga toacgagtgt galaa attalala 5946 aaggggaaat agataaacga gtgtctacat atagaagaag acgtgctatt tgataaatct 59.521, tCtagaaaat aaagtgagtt tggattattt gattttgtta ggtgaataaa. gtgtaaaata 59581 ttgtattitta tgggggaagt tacaaaagat teaatcalata totag tittcta aaagttctgat 5964.1 tggecaaagta tttagatgag tatagoctagg tgagcatttg taagttatac gatticcitcta 59.701 agctittgatt togagtctitt tgttggggtg ttalaattott aaaaaaaaaa acaa tattat 59761 atcc ttettitt taattaaatc. totacgct tta cctttt tatt toatgcaaaa gattgaataa 59821 ttgaacticto tCcactittgg tttgtt tatt Catataaaac caaacagaaa. ataaaaa.cac 5.9881 Cttggtcata accttaatag accgatataa. togggaaagg easaCatala CC talactattta 5994.1 aatgtt tott taacaaaaag. tattgaaact gacctgtttt agttggaa.ca atatgcc tog 60001 cg tgaatatc ggtcCagaCa acaacgctoca aatagagatc. act actic tact aatca Caaag 60.051 caaatcaaga aagctaaaga aaatcaaaat ggctagtgga agtgagcgtg tggtggttgt 6021 cggagtagat gattcagocc atagoctacca tgcacticgag acggctcittg atctosttt tt 6O18. catacctitt aaggcCaaCC cacgattocaa. acttgtcgto cittcacgctic gaCCaaCagc 60241 gaccttcttic Ctcggtgttg Ctggc.ccggg aacggtocgat attataCCaa. tggtggaaga 60301 agatttaaac aagaccg.cgg acttggttaa. galagaagtgc gCCgaagtgt. gotcggctaa 60361 gtCCgttgag atctdgtott tggaggtgat tgaaggagat CCgagaaa.ca taatgttgga 60A21. agccgtggag aga CatCatg catcgagttct tgtcCttggit agcCatggitt atggagctga 60481 CgtCaagagg atgtttittgt ggagcgtCaa gtgttgcgtaa ctatttagot cattaa.gcac 60541 attcCtc.cgt. totgatcatc aagaa.gocta aggatalaa CC tact tctacc aataalacago 60601 aatgaggaac aagacgacgt. gattt togta tatgttacca ttctgtttitc ttalagtaaga 6066 CCCtatictat cgaaCattgt gcatactictt citt.cccala: atct tccatt tCaacgittaa. 60721. tagottagot Caatgtata tactcattgt ata tattoac. tatattaatc gcc toctitctt 60781 tgttgttatta atctoctitac tttitt totgt atatattgca taaaaatgaa gttcttagaa 60841 catttt taca tittatctate atgatcataa. ttgaaaggtt CCaattalaat ttgttctittg 50901 gaaactitg ta. gtaccttgca aagagcttga tCatgtaccala agctgaggta gaaattaaac 60951. Cttatcgtga atgtactata accalatgttg cc.gagaactt gtaagcaatg gCttCag taa 6102 cttct tttitc titt totcagt ttacct tac Cact ttgttga gtCttgtttg Cataataagg 6081 tttgatccga. gottg tagaa gacgc.cactt gtgttt tatg CgaaagaCat taaaac tec 61.141 tatalaccalaa taatcatgtt gctgactgct gaggacttgt aaacaatcga. ttt tagcaac 61.20 ttittct tc ttt totcagt ttacctcitac Cattittatga gtCaagtttg Catalaca agg 61261. tttcaatcc.g agcttgttgta agataa.cact tggtgttctta aatataaaaa. gatccggcaa. 61321 tgttgattitc. cittctalagca a tattt toala tct tcaaact catttata.ca tagittaaCat 61.381. aacCtcCaaa. acataalatat cacagat.cca aaagatcgga taalacataat citctocaaat 61441 aatgatgcgt. tactcitcata ctCtcCagca gttctoraatt ctatoctittca togtocacaat 550 atggaccgtC caatcgg tac CaCCaCagCC accggttcga tgCgatcaaa. CCggCCCaC 5561 CgtCtcCaat gCaaaagagt gtct tccaca aattcactga gCCCttctgag aaagagagaC 6162 acaatgcCaC alactaaaaat tgaactgaaa attgtagg gC tttaccccala agggagcCCt 6168 torcagalaatt CtcgaaggcC CaagaCaago tt
Restrictions on Use Write to th Helpdesk NCBI NLMNH Patent Application Publication Apr. 8, 2004 Sheet 37 of 40 US 2004/0067506 A1
FIGURE 15
Patent Application Publication Apr. 8, 2004 Sheet 38 of 40 US 2004/0067506 A1
FIGURE 16
US 2004/OO67506 A1 Apr. 8, 2004
NOVEL ROOT SPECIFIC PROMOTER DRIVING driving tissue specific gene-expression in plants. It is there THE EXPRESSION OF A NOVEL LRR fore important that Such control elements are continuously RECEPTOR-LIKE KINASE provided and that new control elements with their specific transcriptional features are isolated and characterized. FIELD OF THE INVENTION 0007 Promoter Regulation 0001. The present invention relates to the field of plant 0008 Promoters from both monocots and dicots that have molecular biology, more particularly to the root-specific a root root-preferred expression pattern have already been gene expression in plants. The isolation of a root Specific described (Fowler, Bernhardt, & Tierney 1999; Hwang & operon, comprising a transcriptional regulatory promoter Goodman 1995; Lagarde et al. 1996; Okada et al. 2000; that contributes to root-Specific gene expression and its Okumura et al. 1994; Pysh et al. 1999; Schnelder et al. 1998; operably linked gene encoding a novel Leucin Rich Repeat Vidmar et al. 2000; Wanapu & Shinmyo 1996; Winicov (LRR) receptor-like kinase, is disclosed. Said transcriptional 2000) WO9007001(Baszczynski et al. 2000; Borg et al. regulatory promoter may be used for driving root-specific 1997; Masuda, Sakuta, & Satoh 1999; Webb et al. 2000) (de expression of at least one gene of interest in a transgenic Pater & Schilperoort 1992; Ely, Evans, & Schuch 2000; plant and Said encoded LRR receptor-like kinase gene may Goddemeier, Wulff, & Felix 1998; Kikuchi et al. 1999; be used to alter the features and/or to confer a Selective Suzuki, Fowler, & Tierney 1993; Uribe et al. 1998; Woo & advantage to transgenic plants. Hawes 1997; Xu et al. 1995) (Yamamoto et al. 1991) (Giritch et al. 1997; Keller & Lamb 1989; Lauter 1996; BACKGROUND TO THE INVENTION Olson, Oetiker, & Yang 1995; van der Zaal et al. 1991). Many of them are root-preferred rather then root-specific 0002 Plant Promoters and are expressed at low levels in tissues other than the root. 0003) Initiation of transcription is generally understood These promoters are not Suitable for industrial applications to be the predominant controlling factor in determining where it us of the utmost importance that the heterologous expression of a gene. The transcriptional control elements, product is expressed in the root only. AS an example, ectopic which may interact with DNA binding proteins, are gener expression of cell cycle control elements to obtain growth ally embedded in the Sequence 5'-flanking or upstream of the arrest in Some parts of the root (e.g. lateral roots), should be transcribed gene. These DNA sequence elements promote Strictly regulated to that tissue in order not to interfere with the formation of transcriptional regulatory complexes that the overall growth of the plant. In contrast, other promoters either activate or repress the expression of the gene 3' are strictly and eXclusively confined to root-specific expres downstream of the promoter. Sion and can therefore be valuable for Some applications demanding tight control of the expression. In this group of 0004. The sequences of the promoter vary in length and root-specific promoter Subgroups can be distinguished based base pair composition from gene to gene. Within the on the root-tissue they are expressed in. The HRGPnt3 gene Sequence of the promoter, many regulatory Sequence ele of tobacco for instance is only expressed in the pericycle and ments can be embedded that control the promoter activity. the endodermis of the root, more Specifically in the discrete These specific elements, called DNA-boxes, contribute to Subset of cells, involved in the initiation of the lateral roots the promoter a defined feature or a defined activation (Keller et al. 1989). This makes the promoter of HRGPnt3 pattern. Many of these boxes are recognition sites to which highly specific. Another example is the XSP30 sequence, regulatory transcription factors can bind and are part of a tight control mechanism of the promoter. Numerous plant which is only activated in the xylem of the root. promoter DNA boxes have been described, such as tissue 0009 For persons skilled in the art it is of importance to specific boxes (Chaubet et al. 1996), pyrimidin boxes have at their disposal a wide variety of root-Specific pro (Gubler & Jacobsen 1992), which Influence the level of moters with different root tissue-specificity from which they expression or G-boxes (Dolferus et al. 1994) which reduce can choose to confer a specific feature to the transgenic promoter activity by cold or dehydratation. plant. 0005 Constitutive promoters are promoters that drive the 0010 Many of these root-preferred promoters are not expression of genes in a continuous manner, whereas ubiq only limited to tissue Specific expression, but are also uitous promoters drive the expression of the gene throughout Subjected to a tight control mechanism deciding when the the entire plant. promoter should be active. This decision depends on the 0006 Expression of heterologous DNA sequences in a function and the effects of the gene product that is Switched host is dependent upon the presence of an operably linked on/off by the related promoter. Some gene products are promoter that is also present in that host, whereby the choice Switched on/off at a certain developmental Stage of the plant, of promoter will determine when, where or how Strong the at a certain moment of the cell cycle, in StreSS Situations heterologous DNA sequence will be expressed in the host originating from weather factors, upon injury caused by organism. Frequently those skilled in the art desire to limit pathogens or upon a change in the environmental resources. the expression of the transfected DNA sequence to a certain A typical example is the ARSK1 gene of Arabidopsis time-period (e.g. to a particular phase of plant development), thaliana, that is activated when the roots are exposed to air to a defined tissue of the host (e.g. leaves), or to a certain during growth (dehydration) or by treatment of roots with expression level. Controlling the expression of the heterolo abeisic acid or salt (Hwang et al. 1995). gous genes in transgenic plants is considered to provide 0011. However, for many industrial and agronomic appli Several advantages to the plant over ubiquitous and consti cations, where the aim is to confer a special feature at any tutive expression. There is a broad need in many industrial time of the developmental, environmental or physical State applications for transcriptional control elements capable of of the plant, a constitutive promoter will be needed. For US 2004/OO67506 A1 Apr. 8, 2004 example when the gene product of the transgene is to be the clv1 gen is initiated in the heart Stage of the embryo, isolated and purified from the plant for commercial pur when cotyledonary primordia are apparent, and is indepen pOSes. dent of the STM (SHOOT MERISTEMLESS) activity (Long & Barton 1998). CLAVATA1 is a leucine-rich recep 0012. It is important for the persons skilled in the art to tor-like kinase. The Arabidopsis thaliana genome project have at their disposal a wide variety of promoters with has so far identified over 50 LRR transmembrane receptor different time-control features from which they can chose to Serine/threonine kinases. It can be estimated that the total confer a specific feature to the transgenic plant. Arabidopsis thaliana genome contains in the order of 100 0013 Plant Root Development family members. Other members of the CLAVATA family are ERECTA (ER) (Torii et al. 1996), BRASSINOLIDE 0.014 Root development is an essential determinant of INSENSITVE (BRI) (Li & Chory 1997) and HAESA(HAE) plant growth and crop yield since the root is the main (Jinn, Stone, & Walker 2000), genes which have a function channel to extract nutrients from the environment. Any in plant development. Analogues of the Arabidopsis changes in the root that contribute to more functional organ thaliana CLAVATA genes were also found in soybean tissue will result in higher crop yield. Also any regulatory (Glycine max). They were isolated from the wild type and element that is involved in the root development and whose the fasciating mutant of Soybean and were designated control is Subjected to developmental events can contribute GmCLVA1 and GmCLV1B (Yamamoto, Karakaya, & Knap better yield properties to a transgenic plant. 2000). 0.015 Cells of the shoot and root meristems are mitoti 0020. It is the aim of the present invention to isolate novel cally active and they extend the undifferentiated tissue promoters and genes which are useful for altering features of pattern of the embryo as a pool of cells from which cells can transgenic plants and thus confer Selective advantage to differentiate into several tissues of the adult plant. This transgenic plants. proceSS continues throughout the life span of the plant. It is clear that the root meristem plays a pivotal role in the 0021 All the aims of the present invention have been met formation of Strong roots and therefor also in affecting the by the formulation of the following preferred embodiments growth and crop yield. of the current invention. 0016. In root meristem, the emerging picture is that the SUMMARY OF THE INVENTION integrity of the root meristem is kept by balancing cell proliferation and cell differentiation, and that differentiation 0022. In the present invention a DNA operon was isolated inhibiting Signals originating from the quiescent center are from Arabidopsis thaliana, which comprises a novel root involved (van den Berg et al. 1997). Inhibition of differen Specific promoter, driving a gene encoding a Leucine Rich tiation only occurs in cells that contact the quiescent center, Repeat (LRR) receptor-like kinase. Unexpectedly, the inven which reveals that short-range or contact-dependent Signals tors discovered that the new operon was specifically are involved in this proceSS. However which compounds expressed in roots and more Surprisingly in the meristem of play a role in this process and whether positional cues the main and lateral roots, in the main and lateral root guiding root cell fate are targeted via plasmodesmata or via vascular tissue and in the lateral root primordia. the cell wall remains unknown (van den Berg et al. 1997). 0023 The Surprisingly specific expression pattern, as 0.017. In the shoot meristem more progress has been well as the very Strong expression level, makes the newly made in characterizing molecular components involved in found promoter a very attractive and Selective tool to drive meristem maintenance. Clavata (clv) mutants accumulate root-specific expression of any gene of interest. The ability massive pools of undifferentiated cells in the central Zone of to target meristem cells of the root makes this promoter shoot and floral meristem. The CLAVATA genes show a clearly distinct from the root-Specific promoters presently shoot and floral meristem specific expressionpattern. Root known in the art. Another Surprising aspect of the invention meristems of clv-mutants are unaffected (Clark, Running, & is the identification of the new LRR receptor-like kinase Meyerowitz 1993). The CLAVATA loci (CLV1, CLV2 and gene, which is the first gene identified in roots showing CLV3 appear to promote the transition toward differentia homology with a clavata-gene. This gene is specifically tion of cells at the shoot and floral meristems, and/or to expressed in roots in a Specific developmental Stage of the restrict the proliferation of cells at the center of these root cells (namely in the meristematic phase) and can meristems (Clark et al. 1993). CLV1 acts with CLV3 and therefore contribute agronomic interesting features to a WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) transgenic plant when transfected herein. Therefore the to maintain the integrity of the central Zone which acts as a root-specific promoter is further denominated as ROOT reservoir of Stem cells. A model for Stem-cell maintenance CLAVATA HOMOLOG 1 promoter (RCH1 prom) and the in the root meristem and in the shoot meristem is depicted LRR receptor-like kinase as RCH1. in FIG. 1. 0024. Accordingly, the invention embodies an isolated DNA sequence with nucleotide sequence as given in SEQID 0018 CLV1 Homologs and CLV1-Redundancy NO 1 (FIG. 5) comprising a novel root specific regulatory 0019. The CLAVATA1 gene resides on the Arabidopsis promoter Sequence with nucleotide Sequence as given in thaliana chomosome 1. Clv1 mRNA is found in the L3 SEQ ID NO 2 (FIG. 6) or SEQ ID NO 18 (FIG. 13), and tunica cell layer of the central Zone and encodes a protein an isolated DNA sequence with nucleotide Sequence as with leucine-rich repeats (LRR, of a type found in a number given in SEQ ID NO 3 (FIG. 8), encoding a novel ROOT of proteins, notably the products of Several plant-pathogen CLAVATA HOMOLOG 1 gene, with amino acid sequence resistance genes), a putative serin/threonin kinase domain given in SEQ ID NO 4 or SEQ ID NO 19 (FIG. 9). and a predicted extracellular receptor domain. Expression of Furthermore the invention embodies a method for modifying US 2004/OO67506 A1 Apr. 8, 2004
cell fate and/or plant development and/or plant morphology SEQ ID NO 4, SEQ ID NO 19 or to a nucleic acid and/or biochemistry and/or physiology comprising the Sequence as defined in (a) to (d), modification of expression in particular cells of the root of a particular transgene operably linked to the RCH1 promoter 0033 (g) nucleic acid encoding a protein as given in Sequence of the present invention. Provided in the current SEQ ID NO 4, SEQ ID NO 19 or as defined in (a) invention are methods to effect expression of any gene of to (d), which is diverging due to the differences interest in particular cells of the plant root. The present between alleles encoding a protein invention also relates to a method to effect expression of a 0034 (h) nucleic acid encoding a fragment of a ROOT CLAVATA HOMOLOG 1 protein or a homologue or protein encoded by a DNA sequence as given in SEQ a derivative thereof in a plant cell, tissue or organ. The ID NO 1 or SEQID NO3 or encoding a fragment of present invention clearly extends to any plant produced by a protein encoded by a nucleic acid as defined in any the inventive methods described herein. one of (a) to (g), wherein said fragment comprises DETAILED DESCRIPTION OF THE the sequence as represented in SEQ ID NO 5, and, INVENTION 0035) (k) nucleic acid encoding a protein as defined 0.025 One of the technical problems underlying the in SEQ ID NO 4, SEQ ID NO 19 or nucleic acid as present invention is to provide regulatory elements that are defined in any one of (a) to (h), Said nucleic acid active in the root only and that show no expression in other interrupted by intervening DNA sequences. tissues. Additional to the highly Selective and tissue-specific 0036) The sequence as depicted in SEQ ID NO 1 is part regulation of a promoter, other features Such as very Strong of a Sequence deposited under the Genbank Accession activity, continuous activity, and termination of activity at a number ABO 17061, which represents the entire Arabidopsis certain developmental Stage of the cells, would contribute an thaliana chromosome 5. The nucleic acid of the invention is unique combination of regulatory elements to specifically not the nucleic acid with the Sequence as deposited under the regulate the expression of a gene of interest in the roots of Genbank Accession number ABO17061 (FIG. 14). Further a transgenic plant, thereby providing a means for modifying more, in a preferred embodiment, the present invention cell fate and/or plant development and/or plant morphology relates to an isolated nucleic acid comprising a novel root and/or biochemistry and/or physiology. Also the isolation of Specific promoter Sequence, termed RCH1 prom, isolated root-specific DNA sequences encoding proteins that could from Arabidopsis thaliana and consisting of the nucleic acid play a role in root development is envisaged for use in as represented in SEQ ID NO 2 or SEQ ID NO 18, or a modification of cell fate and/or plant development and/or functional part of Said Sequence, which is able to regulate plant morphology and/or plant biochemistry and/or plant gene expression in a root specific manner as SEQ ID NO 2 physiology. or SEQ ID NO 18 itself. More specifically, said isolated 0026. In a first embodiment, the present invention relates DNA sequence provides a novel type of a root-specific to an isolated nucleic acid comprising a transcriptional promoter, which differs from other known root-specific regulatory root promoter Sequence and/or a nucleic acid promoters in the fact that it is unexpectedly active in only Sequence encoding a novel LRR receptor-like kinase (pro distinct cells of the root, namely the meristem of the main tein) or an immunologically active and/or functional frag and the lateral root, as well as in the vascular tissue of the ment of Such a regulary element or protein (kinase) Selected main and lateral root and in the lateral root primordia. from the group consisting of: 0037. The isolation of the root specific promoter RCH1 0027 (a) nucleic acid comprising at least part of the of the present invention is described in Example 1. DNA sequence as given in any of SEQ ID NOS 1, 2, 0038 Another aspect of the invention is the isolation and 3 or 18, or the complement thereof, Sequencing of a root-specific Leucin Rich Repeat (LRR) receptor-like kinase gene from root tissue of Arabidopsis 0028 (b) nucleic acid comprising the RNA thaliana and is also described in Example 1. The nucleotide Sequences corresponding to at least part of any of sequence is listed in the current specification as SEQID NO SEQ ID NOS 1, 2, 3 or 18, or the complement 3 and illustrated in FIG. 8. This sequence is present as part thereof, of the Sequence deposited under the GenBank Accession 0029 (c) nucleic acid specifically hybridizing, pref number ABO 17061, which represents the entire Arabidopsis erably under Stringent conditions, with the nucle thaliana chromosome 5. otide Sequence as defined in (a) or (b), 0039. The novelty of the RCH1 promoter sequence rela 0030) (d) nucleic acid encoding a protein compris tive to known root-Specific promoters is Substantiated by its ing the amino acid sequence as given in SEQ ID NO unique expression pattern in plant roots. The analysis of the 4, SEQ ID NO 5 or SEQ ID NO 19, RCH1 expression patterns was done using a RCH1 prom GUS hybrid operon that was transformed to Arabidopsis 0031 (e) nucleic acid which is degenerated as a thaliana as outlined in Example 2. Analogous experiments result of the genetic code to a nucleotide Sequence with the RCH1 prom-GUS hybrid operon were performed to encoding a protein as given in SEQ ID NO 4, SEQ establish the expression pattern of RCH1 in embryo's ID NO 19 or to a nucleotide sequence as defined in (Example 8 and FIG. 15). (a) to (d), 0040. The RCH1 promoter is active in the meristem of 0032 (f) nucleic acid which is diverging due to the the main and the lateral roots, as well as in the vascular differences in codon usage between the organisms to tissue of the main and the lateral root and in the lateral root a nucleotide Sequence encoding a protein as given in primordia. The promoter of the present invention is the first US 2004/OO67506 A1 Apr. 8, 2004
known promoter that regulates a CLAVATA HOMOLOG root-specificity to other promoters, but a Second Sequence is gene in roots, and is clearly distinct from other described used to confer an additional regulatory feature to the RCH1 CLAVATA-operons which are all active in the shoot and promoter. floral meristem. The present data thus clearly distinguish 0050. In another embodiment said second transcriptional RCH1 prom from other plant root-specific promoters, which regulatory Sequence is a promoter Sequence not normally are not expressed in the meristem of the main and lateral exhibiting root-specificity, thereby resulting in a hybrid root, in the vascular tissue of the main and lateral root and in the lateral root primordia. promoter with root-Specificity. 0051. Accordingly, another embodiment of the present 0041). From the results obtained by the RCH1::GFP invention is to confer root-specificity, root-meristem-speci expression Studies in root it was even more clear that the ficity, and root-Vascular-tissue-specificity and/or root abun RCH1 promoter is strongly active in the endodermis, cortex, dant gene expression to other promoter Sequences. This can epidermis, lateral root cap, in Short in the “division Zone'. be achieved by fusing elements of the here-disclosed RCH1 The RCH1 promoter is also active but in a lower manner, in promoter to the promoter Sequence of interest preferably the the quiescent center and in the vascular tissue. widely used ubiquitin promoter. Such modifications can be 0.042 A further characteristic of the RCH1 promoter is its achieved by routine experimentation by those skilled in the very high expression level. Based on the incubation time of art. the RCH1 prom-GUS hybrid operon transformed plant with 0052. It should be further understood that the invention the GUS Substrate (1 hour) and the very high output signal preferably relates to any of the isolated nucleic acids as in the root of that plant (Example 2, FIG. 7D), it is clear for described above which is DNA, cDNA, genomic DNA, persons skilled in the art that the RCH1 promoter is very synthetic DNA or RNA wherein T is replaced by U. Strong. 0053. The invention also relates to a nucleic acid mol 0.043 A further characteristic of the present invention is ecule of at least 15 nucleotides in length hybridizing Spe that the activity of the RCH1 promoter in the meristem cells cifically with any of the nucleic acids of the invention. The Stops when these cells Start to differentiate and to elongate invention also relates to a nucleic acid molecule of at least to become cells of Specified root tissue other than root 15 nucleotides in length specifically amplifying a nucleic vascular tissue. Surprisingly the promoter is activated again acid of the invention. in cells of the pericycle that form lateral root primordia. This expression is extended in the meristem of the lateral root, but 0054 Further, the invention relates to a vector compris again, the cells that differentiate into lateral root tissue other ing a nucleic acid of the invention. Said vector may be an than vascular tissue do not retain RCH1 promoter activity. expression vector wherein the (said) nucleic acid sequence encoding a novel LRR receptor-like kinase or an immuno 0044) Functional parts of the promoter of the invention logically active and/or functional fragment thereof, is oper will have at least one of these characteristics. ably linked to one or more control Sequences allowing the 0.045 According to a further embodiment, the present expression in prokaryotic and/or eukaryotic host cells. invention relates to an isolated nucleic acid comprising a 0055. The invention also relates to a vector comprising at transcriptional regulatory root promoter comprising at least least part of a nucleic acid according to the invention and part of a sequence as given in SEQ ID NO 1, 2 or 18. wherein a transcriptional regulatory root promoter of the invention or functional part(s) thereof, is operably linked to 0.046 According to another embodiment, the present one or more genes of interest. invention relates to an isolated nucleic acid as represented in SEQ ID NO 2 or 18 wherein said transcriptional regulatory 0056. The invention also relates to a host cell containing root promoter is operably linked to Said nucleic acid a nucleic acid molecule of the invention or a vector of the Sequence encoding a novel LRR receptor-like kinase or to an invention wherein Said nucleic acid or vector has been immunologically active and/or functional fragment thereof. introduced by transfomation, transfection or infection. Pre ferred host cells that are part of the invention are bacterial, 0047 According to the invention, distinctive fragments insect, fungal, plant or animal cells. of the newly identified promoter, which contribute specific features to Said promoter, can be fused to another nucleic 0057 Therefore, in a preferred embodiment, the inven acid Sequence (e.g. a promoter) in order to alter the Sequen tion relates to an isolated nucleic acid as represented in SEQ tial and/or regulatory features of the latter. ID NO 3 encoding a LRR receptor-like kinase characterized by an amino acid sequence as given in SEQ ID NO 4 or 19. 0.048. The invention thus relates to an isolated nucleic More Specifically, Said isolated nucleic acid encodes a plant acid having transcriptional regulatory root Specific, root ROOT CLAVATA HOMOLOG 1 (RCH1) of a novel type meristem specific or root-Vascular-tissue Specific promoter which is unexpectedly expressed in roots. Said novel plant activity comprising at least part of the DNA sequence as RCH1 shows significant homology with CLAVATA 1, a given in any of SEQ ID 1, 2 or 18 and a second transcrip protein involved in the maintenance of floral and Shoot tional regulatory Sequence. meristem cells. 0049. In one embodiment, said second trancriptional 0058 An initial comparative amino acid sequence regulatory Sequence is for instance an inducible box or homology search using the BLASTP 2.0.8 software (Alts Sequence, which is placed within the RCH1 promoter, chulet al. 1997) revealed that most significant alignments of wherein the function of the RCH1 promoter is not hampered RCH1 protein are produced with receptor-like protein but instead can be regulated, for instance by induction. In the kinases, more specifically with leucine rich receptor-like latter embodiment, the RCH1 promoter is not used to confer kinases such as CLAVATA1 (CLV1). Furthermore, the most US 2004/OO67506 A1 Apr. 8, 2004 homologous protein with a known function of this list, is the 0068 (b) a nucleic acid encoding a protein as given CLAVATA 1 receptor-like kinase from Arabidopsis thaliana. in SEQID NO 4 or 19 or encoding a fragment of said Therefore the novel gene was denominated a ROOT protein, wherein Said fragment comprises the CLAVATA HOMOLOG 1 gene (RCH1). sequence as represented in SEQ ID NO 5, and 0059 An amino acid sequence alignment of the RCH1 0069 (c) a nucleic acid encoding a protein with an protein and CLAVATA 1 from A. thaliana is shown in FIG. amino acid Sequence which is at least 60%, prefer 10. Calculation of the percentage of identical amino acids ably at least 65%, 70%, 75%, 80%, 85%, more residues between RCH1 and CLV1 teaches that RCH1 is preferably at least 90% or 95% identical to the 39% identical to CLV1. Additional to 39% of identical protein as given in SEQ ID NO 4 or 9, wherein said amino acids, there is 49% of the amino acids that belong to amino acid Sequence comprises the Sequence as the same class of amino acids and therefore have the same represented in SEQ ID NO 5, characterised in that physical and chemical properties. Said nucleic acid encodes a novel LRR receptor-like kinase protein or an immunologically active and/or 0060. The classification of RCH1 as a novel type of functional fragment of Such a protein, and further CLAVATA is documented further in FIGS. 11 and 12. provided that Said nucleic acid is not one of the 0061 The conclusions we can draw from FIG. 11 is that nucleic acids as deposited under the GenBank Acces based on the sequence, RCH1 is closer related to CLV1 Sion numbers ABO 17061 or AO966419. compared to other known CLV1 homologues, Such as 0070 The present invention also relates to an isolated ERECTA, BRASSINOLIDE INSENSITIVE and HAESA. LRR receptor-like kinase (protein) comprising one of the 0062) The fact that the novel LRR receptor like protein polypeptides Selected from the group consisting of: described in the present invention is rather a root clavata 1 0071 (a) a polypeptide as given in SEQID NO 4 or homologue then another kind of LRR receptor-like kinase SEQ ID NO 19, (e.g. resistance genes) is illustrated in FIG. 12 in which the evolutionary relationship between clavata homologues and 0072 (b) a polypeptide comprising the amino acid resistance genes is shown. The resistance genes (RPS5, sequence as given in SEQ ID NO 5, and, RPS2, RPP8, RPM1, RPS4, N, RPP5) are described in 0073 (c) a polypeptide comprising the sequence “Meyers BC, Dickerman AW, Michelmore RW, Sivara represented in SEQ ID NO 5 and encoded by a makrishnan S, Sobral B W, Young N D. Plant disease nucleic acid as given in SEQ ID NO 1 or 3, resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding Super family. 0074 or a homologue or a derivative of said protein, or a fragment or an immunologically active and/or functional Plant J. 1999 November; 20(3):317-32". fragment thereof, provided that Said homologue is not the 0063) The present invention relates to a ROOT amino acid Sequence as described under the GenBank CLAVATA HOMOLOG 1 gene that differs from other Accession number BAB10317.1. known CLAVATA-like genes, in the fact that it is active in the root meristem, whereas the other known CLAVATA 0075 With the expression “homologue” when referring genes and CLAVATA homologs (like ERECTA, BRASSI to homologues of the LRR receptor-like kinase (protein), a NOLIDE INSENSITIVE and HAESA) are only active in the polypeptide is meant which is at least 65% identical to the shoot and floral meristem. amino acid sequence of RCH1 as represented in SEQID NO 4 or 19. 0064. According to another embodiment, the invention 0076 More preferred, the invention relates to a protein also relates to nucleic acids encoding a functional plant LRR consisting of an amino acid Sequence as given in SEQ ID receptor-like kinase comprising one or more protein regions, NO 4 or SEQ ID NO 19. More specifically, the present distinguishing Said plant LRR receptor-like kinase from invention relates to a root CLAVATA homologue or a those LRR receptor-like kinases known in the art. functional homologue thereof. A preferred fragment of Said 0065. The inventors found that the predicted amino acid protein is represented in SEQ ID NO 5. Sequence (BAB10317.1, receptor protein kinase-like pro 0077. A further embodiment of the invention comprises tein) corresponding to the database entry with accession homologues, derivatives, immunologically active and/or number ABO17061 (A. thalinana chromosome 5), was functional fragments of the LRR receptor-like kinase (pro missing a part of the amino acid sequence (namely, 25 amino tein) according to the invention, and proteins comprising acids as represented in SEQ ID NO. 5 of the present patent Said homologues, derivatives, immunologically active and/ application). This extra stretch of 25 amino acids was or functional fragments of Said LRR receptor like kinase identified by the cloning and the Sequencing of the corre (protein). sponding cDNA of the present invention. Therefore, the predicted prior art Sequence is believed not to represent a 0078. As such, the present invention also relates to an functional enzyme. isolated polypeptide comprising the Sequence represented in SEQ ID NO 5 encodable by a nucleic acid molecule of the 0.066 The invention thus relates to an isolated nucleic invention as defined above, or a homologue or a derivative acid Selected from the group consisting of thereof, or a fragment or an immunologically active and/or 0067 (a) a nucleic acid consisting of at least part, functional fragment thereof. preferably at least a functional part, of the DNA 0079. In a more preferred embodiment, the invention sequence as given in SEQ ID NO 1 or 3, or the relates to a polypeptide, encodable by a nucleic acid mol complement thereof, ecule of the invention and which has an amino acid Sequence US 2004/OO67506 A1 Apr. 8, 2004
as given in SEQ ID NO 4 or SEQ ID NO 19 or comprising preferably Selected from the group consisting of Seeds, an amino acid sequence as given in SEQ ID NO 5, or a leaves, fruits, Stem cultures, rhizomes, tubers and bulbs. homologue or a derivative thereof, or an immunologically 0087. The invention further extends to the progeny active and/or functional fragment thereof. derived from any of the plants or plant parts described 0080) Any of said proteins can be produced in a biologi above. cal System, e.g. a cell culture. Alternatively any of Said 0088. The present invention is applicable to any plant, in proteins is chemically manufactured e.g. by Solid phase particular a monocotyledonous plants and dicotyledonous peptide Synthesis. Said proteins or fragments thereof can be plants including a fodder or forage legume, ornamental part of a fusion protein as is the case in e.g. a two-hybrid plant, food crop, tree, or shrub Selected from the list com assay which enables e.g. the identification of proteins inter prising Acacia spp., Acer spp., Actinidia spp., AeSculus spp., acting with the LRR receptor-like kinase according to the Agathis australis, Albizia amara, Alsophila tricolor, Andro present invention. pogon spp., Arachis spp., Areca catechu, A.Stelia fragrans, 0081. Therefore, according to another embodiment, the AStragalus Cicer, Baikiaea plurijuga, Betula spp., Brassica invention also relates to a method for producing a polypep spp., Bruguiera gymnorrhiza, Burkea africana, Butea fron tide or a protein of the invention comprising culturing a host dosa, Cadaba farinOSa, Calliandra spp., Camellia Sinensis, cell further Specified above under conditions allowing the Canna indica, Capsicum spp., Cassia spp., Centroema pube expression of the polypeptide and recovering the produced Scens, Chaenomeles spp., Cinnamomum cassia, Coffea ara polypeptide from the culture. bica, Colophospermum mopane, Coronillia varia, Coto neaster Serotina, Crataegus spp., Cucumis spp., CupreSSuS 0082 The proteins or fragments thereof obtained by a spp., Cyathea dealbata, Cydonia Oblonga, Cryptomeria method of the invention are furthermore useful e.g. to japonica, Cymbopogon spp., Cynthea dealbata, Cydonia modulate the interaction between a LRR receptor-like kinase oblonga, Dalbergia monetaria, Davallia divaricata, Des according to the invention and a ligand of the receptor and/or modium spp., DickSOnia SquarOSa, Diheteropogon amplect other identified interacting protein partners. Chemically ens, Dioclea spp., DolichoS spp., Dorycnium rectum, Echi Synthesized peptides are particularly useful e.g. as a Source nochloa pyramidalis, Ehrartia spp., Eleusine coracana, of antigens for the production of antisera and/or antibodies. EragreStis spp., Erythrina spp., Eucalyptus spp., Euclea 0.083. The current invention thus furthermore encom Schimperi, Eulalia villosa, Fagopyrum spp., Feiloa Sellowi passes antisera and/or antibodies Specifically recognizing ana, Fragaria spp., Flemingia spp., Freycinetia banksii, the LRR receptor-like kinase according to the invention or Geranium thunbergii, Ginkgo biloba, Glycine javanica, immunologically active parts or epitopes thereof. Said anti Gliricidia spp., Gossypium hirsutum, Grevillea spp., Gui Sera and/or antibodies are useful in many areas related to the bOurtia ColeOSperma, Hedysarum spp., Hemarthia altissima, invention including: (i) identification in any organism, pref Heteropogon contortus, Hordeum vulgare, Hyparrhenia erably plants, of other LRR receptor-like kinase and their rufa, Hypericum erectum, Hyperthelia dissoluta, IndigO genes according to the invention; (ii) quantification of incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Synthesis in organisms and/or recombinant organisms of the Lettuca spp., Leucaena leucocephala, LOudetia Simplex, LRR receptor-like kinase according to the invention; (iii) LOtOnus baineSii, Lotus spp., Macrotyloma axillare, Malus purification of the LRR receptor-like kinase according to the spp., Manihot esculenta, Medicago Sativa, MetaSequoia invention; (iv) immunoprecipitation of the LRR receptor glyptoStroboides, Musa Sapientum, Nicotianum spp., Ono like kinase according to the invention e.g. as a way to brychis spp., Omithopus spp., Oryza spp., Peltophorum identify other protein partners complexing with Said LRR africanum, Pennisetum spp., Persea gratissima, Petunia receptor-like kinase, (v) immunolocalization of the LRR spp., Phaseolus spp., Phoenix canariensis, Phormium COOki receptor-like kinase according to the invention which is anum, Photinia spp., Picea glauca, Pinus spp., Pisum Sati expressed in an organism or a recombinant organism. vum, Podocarpus totara, Pogonarthda fleckii, Pogonarthria Squarrosa, Populus spp., PrOSopis cineraria, Pseudotsuga 0084. The invention also relates to a method for the menziesii, Pterolobium Stellatum, Pyrus communis, Quercus production of transgenic plants, plant cells or plant tissues spp., Rhaphiolepsis umbellata, RhopaloStylis Sapida, Rhus comprising the introduction of a nucleic acid molecule of the natalensis, Ribes grOSSularia, Ribes spp., Robinia pseudoa invention in an expressible format or a vector as described cacia, Rosa spp., Rubus spp., Salix spp., Schyzachydium earlier in Said plant, plant cell or plant tissue. Sanguineum, SciadopityS verticillata, Sequoia Sempervirens, 0085. The invention also relates to a method as described Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., above further comprising regenerating a plant from Said Sporobolus fimbnatus, Stiburus alopecuroides, Stylosanthos plant cell. humilis, Tadehagi spp., Taxodium distichum, Themeda trian dra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vac 0.086. In a further embodiment, the invention also relates to a transgenic plant cell comprising a nucleic acid Sequence cinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, of the invention which is operably linked to regulatory ZantedeSchia aethiopica, Zea mayS, amaranth, artichoke, elements allowing transcription and/or expression of Said asparagus, broccoli, brussel Sprout, cabbage, canola, carrot, nucleic acid in plant cells or obtainable by one of the cauliflower, celery, collard greens, flax, kale, lentil, oilseed methods described above. Preferably, in said transgenic rape, okra, Onion, potato, rice, Soybean, Straw, Sugarbeet, plant cell one of the nucleic acids of the invention is stably Sugar cane, Sunflower, tomato, Squash, and tea, amongst integrated into the genome of Said plant cell. The invention others, or the Seeds of any plant Specifically named above or further relates to a transgenic plant or plant tissue compris a tissue, cell or organ culture of any of the above Species. ing Said plant cells and to a harvestable part of Said plant or 0089. Yet according to another embodiment the present tissue. Said harvestable part of Said plant or tissue is invention relates to a method for conferring root-Specificity US 2004/OO67506 A1 Apr. 8, 2004 and/or root-meristem-specificity and/or root-Vascular-tis environmental remediation comprising the expression of a Sue-Specificity and/or root-endodermis-specificity and/or gene(s) of interest under the control of a transcriptional root-cortex-specificity and/or root-epidermis-specificity regulatory root promoter as defined earlier. and/or lateral-root cap-specificity and/or abundant expres Sion in roots to other promoter Sequences comprising the 0096. In conclusion the present invention provides a fusion of at least part of the DNA sequence as given in SEQ method for broadening the range of Soils or environments in ID NO 1, 2 or 18 to a (second) transcriptional regulatory which a plant may thrive or that can be used for agriculture. promoter Sequence normally not exhibiting root-Specificity. 0097 Plant root development is an essential determinant 0090 The invention further relates to method for root of plant growth and crop yield, So that yield could be Specific expression of a gene(s) of interest comprising enhanced by induced changes in expression of root-specific operably linking of said gene(s) of interest to a transcrip regulatory factors. Cyclin D for example, which promotes tional regulatory root-specific promoter comprising at least the cell division, can be expressed under the root-specific part of the nucleic acids as given in SEQ ID NO 1, 2 or 18 control of the current invention and hence contribute to as defined above, and possibly in combination/or a (Second) increased growth rate and crop yield. Also genes that are transcriptional regulatory promoter Sequence not normally involved in biosynthesis and perception of phytohormones exhibiting root-Specificity, as defined earlier. like auxins, cytokinines, ethylene, abscisic acid and gibe berellins can be used to enhance root growth and develop 0.091 The invention further relates to a method for modi ment. However, the root-Specific expression of these genes fying cell fate and/or plant development and/or plant mor might be pivotal for the overall welfare of the transgenic phology and/or biochemistry and/or physiology comprising plant. the modification of expression in the meristem of the main and the lateral roots, or in the vascular tissue of the root, or 0098. It is known that the constitutive and ubiquitous in the lateral root primordia of a gene(s) of interest operably expression of the cytokinine oxidase in Arabidopsis thaliana linked to a transcriptional regulatory root promoter as leads to the promotion of an enlarged root System and defined earlier. enhanced root biomass, but also to diminished shoot growth. 0092. The ability to drive tissue-specific expression of To overcome this problem the cytokinin Oxidase can be any gene of interest in plants is considered to be of great operably linked to the promoter of the present invention as agronomic importance. This art can be used to exclude described in example 6, which will enhance root growth expression in eatable parts of the plant, which averts the without negatively affecting shoot growth. This enhanced uptake of ectopic components. Alternatively the expression root growth in the plant can be used for bioremidiation or of the transgene can be restricted to the relevant tissues of phytoremediation. the plant So the total organism does not spend too much 0099 Also the said gene of interest, that can be operably energy in Superfluous production of the foreign component. linked to the promoter of the present invention, can be a root This technique can also reduce potential yield loSS by transcription factor, Such as Alfin. Alfin binds to promoter limiting the expression of Some pernicious, yet useful agro fragments of predominantly root-Specific and Salt inducible nomic genes, to specific tissueS only. Said genes of interest, genes and its activity has major implications for crop plant which can be expressed in a root-Specific manner under the yield (Winicov. 2000) (Winicov & Bastola 1999)). In con control of the present invention can also be involved in clusion the present invention provides a method for enhanc phytoremediation, enhanced root-growth (like cell cycle ing plant growth and crop yield. genes or cytokinin oxidase) or disease tolerance. These methods are more elaborated in detail below. 0100. It is also clear for the skilled artisan that the promoter of the present invention provides a useful tool to 0093. The invention described here provides a new tran be used for the introduction of resistance to Soil-borne Scriptional regulatory element which can be operably linked pathogen attack to plants, a widespread target for genetic to a gene effecting and/or modifying root metabolites which improvement of crop plants. Certain disease-inducing can be involved in phytoremediation. Examples are genes microorganisms attack the belowground plant tissue and any involved in the production of citric and maleic acids that can genetic modification to contribute resistance to Such organ be Secreted by the plant and Subsequently form complexes isms will require expression of the resistance-gene in the with the aluminium in the Soil. Root Specific expression of roots. To overcome this problem, the coding region that is the aluminium-resistance gene that give rise to Said metabo operably linked to the promoter of the present invention lites can therefore allow plants to grow on acid Soils, like preferably encodes a protein which is toxic to root-attacking those of South-eastern United States, Central and South organisms and more preferably the protein is an insecticidal America, North Africa and parts of India and China, where endotoxin of Bacillus thuringiensis. Similarly to the above the aluminium is Set free as an ion that poisons plant roots. mentioned pathogens, there are pests attacking plants. Cer 0094. Alternatively, the promoter of the present invention tain insect species attack green leaf tissue, whereas other, for also provides a tool to be used in a strategy for environ example coleoptera, attack the roots. Corn root worm mental remediation. Accordingly, a metal-resistance gene (Diabrotica undecimpunctata howardi Barber) for example such as the TaPCS1 or the CAD1 gene, contributing cad is a particularly difficult pest to control or to eradicate. It mium tolerance, can be expressed Specifically in roots to attacks the plant below the Soil line, where insecticides are enable enhanced growth of transgenic plants on Soils con difficult or impossible to apply effectively. Com root worm taminated with cadmium. The metals can consequently be can be eradicated by root-specific expression of the Cry III extracted from the Soil and Stored in the plant material, gene, which produces a component, Specifically toxic for which could then be harvested and incinerated. coleoptera. Also root-specific expression of the limonene 0.095 Therefore according to preferred embodiments, the cyclase gene in combination with GPPP synthetase would be present invention relates to a method for phytoremidiation or larvicidal. Another pest, nematodes, that penetrate the root US 2004/OO67506 A1 Apr. 8, 2004
and form root-knots or cysts, which cause Severe damage to 0107 The promoter of the present invention drives the crops throughout the world, could be treated with the Arabidopsis thaliana gene ROOT CLAVATA HOMOLOG 1 root-specific expression of nematode resistance genes Such (RCH1). Since the clavata-genes are key role players in as Hs1P'', Ni-1 and Gpa2. shoot-meristem maintenance, perSons skilled in the art may recognize the possible involvement of the RCH1 operon in 0101. In conclusion the invention disclosed here provides root meristem maintenance. Therefor one could assume that a method for conferring enhanced resistance to pathogens to the control of this promoter is influenced by Signals that a transgenic plant which pathogens attack the belowground promote meristem maintenance. This might be an advantage plant tissue comprising the expression of a gene(s) of when aiming to Stimulate the root meristem-formation, interest under the control of a transcriptional regulatory which eventually could result in more roots, higher uptake root-specific promoter as defined earlier. of resources and in higher crop yield. 0102) In yet another embodiment of the invention, the 0108. Therefore, according to preferred embodiments, invention provides a method to enhanced freezing tolerance the invention relates to methods for Stimulating meristem comprising the expression of a gene(s) of interest under the formation and/or maintaining root meristem comprising the control of a transcriptional regulatory root-Specific promoter expression of a gene that influences the proliferation and/or as defined earlier. Preferred is the overexpressing of the differentiation of the root merSiteme under the control of a Arabidopsis thaliana DNA binding factors CBF1 and transcriptional regulatory root-specific) promoter of the DREB1A that induce cold-regulated (COR) genes (Jaglo invention. Ottosen et al. 1998). However, constitutive expression of 0109 The present invention therefore relates to a method these genes in all organs of the plant led to growth retarda for Stimulating root meristem formation or for root-mer tion under normal growth conditions. Root-specific expres isteme maintenance comprising the expression in particular Sion of these freezing-tolerance genes under control of the cells, tissueS or organs of a plant, of a nucleic acid encoding promoter Sequence of the present invention can make the an LRR receptor-like kinase, for instance the LRR receptor plant tolerant to cold temperatures of the Soil without like kinase of the invention, wherein optionally said nucleic affecting the growth of the plant. This technique can have acid is operably linked to a plant-operable promoter important agronomic potential Since lots of Soils are inac Sequence, for instance Said plant-operable promoter being cessible for crop production because of presence of perma the transcriptional regulatory root promoter as defined ear nently frozen layers in the Soil. lier. 0103) Alternatively, other genes of interest can tolerize 0110 AS mentioned Supra, the combined gene expression the transgenic plant for other weather conditions like data and homology to CLAVATA 1 indicate that the novel drought (e.g. oat arginin decarboxylase gene) and high Salt plant LRR receptor-like kinase of the invention, RCH1 conditions (e.g. MtD gene). In conclusion, the present (SEQ ID No. 3, 4, 19), is very likely to exert a function that invention provides a method to contribute to a transgenic is involved in the development and/or maintenance of the plant StreSS-tolerance. root meristem. AS the root is the main plant organ for 0104 Further advantages to root-preferred gene expres nutrient uptake from the environment, enhanced root for Sion include the production of useful proteins in an industrial mation could eventually lead to overall growth enhancement Setting. Light-sensitive proteins may be Synthesized in root and better yield. Therefore preferred genes to be expressed tissue Such that Said proteins are not exposed to light. in the methods for Stimulating meristem formation and/or Therefore the present invention also relates to a method for for root meristeme maintenance are genes encoding the the production of light-sensitive proteins comprising the polypeptide(s) of the invention. expression of a gene encoding Said light-sensitive protein 0111. The present invention also relates to a method for under the control of a transcriptional regulatory root-Specific enhancing root formation and/or root growth comprising the promoter of the invention. expression in particular cells, tissueS or organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase, for 0105. Also considered as an embodiment of the invention instance the LRR receptor-like kinase of the invention, is the use of the RCH1 promoter for applications based on wherein optionally said nucleic acid is operably linked to a the inhibition of expression of native DNA sequences within plant-operable promoter Sequence, for instance Said plant the plant's root to achieve a desired phenotype resulting operable promoter being the transcriptional regulatory root from the Silencing of the native gene. In this case, Such promoter as defined earlier. inhibition might be accomplished with transformation of the 0112 The present invention therefore relates to a method plant to comprise a transcriptional regulatory root-Specific for enhancing overall growth and yield comprising the promoter of the present invention operably linked to e.g. an expression in particular cells, tissueS or organs of a plant, of antisense nucleotide Sequence to the gene of interest, a gene a nucleic acid encoding an LRR receptor-like kinase, for Silencing construct or a ribozyme. instance the LRR receptor-like kinase of the invention, 0106 Those skilled in the art will be aware that plants wherein optionally said nucleic acid is operably linked to a Secure the formation of organs throughout their life span by plant-operable promoter Sequence, for instance Said plant developing and maintaining a collection of Stem cells termed operable promoter being the transcriptional regulatory root the meristem. The promoter of the present invention offers promoter as defined earlier. the opportunity to deliver a certain protein Specifically to 0113. The present invention also relates to a method for these cells. For example overexpression of genes that influ enhancing overall growth and yield comprising the expres ence the proliferation and/or the differentiation of the root Sion of a protein of the invention under the control of a meristem could contribute to a higher turnover of the transcriptional regulatory root (or root-specific) promoter of meristem cells and therefor enhance the root architecture. the invention. US 2004/OO67506 A1 Apr. 8, 2004
0114. Also in yet other embodiments of the invention, 0123 a) providing a two-hybrid screening system methods are provided for modifying cell fate and/or plant wherein a polypeptide or protein of the invention and development and/or plant morphology and/or biochemistry a protein interacting with Said (LRR receptor-like and/or physiology comprising the modification of expres kinase) polypeptide or protein or an interacting pro Sion in particular cells, tissueS or organs of a plant, of a tein obtainable by a method of claim as claimed nucleic acid encoding an LRR receptor-like kinase, for above are expressed, instance the LRR receptor-like kinase of the invention and defined above, wherein optionally Said nucleic acid is oper 0124 b) interacting said compound with the com ably linked to a plant-operable promoter Sequence, for plex formed by the expressed proteins as defined in instance Said plant-operable promoter being the transcrip a), tional regulatory root promoter as defined earlier. 0125 c) detecting a second complex, wherein the 0115 The present invention further relates to a method to presence of Said Second complex identifies a com confer pathogen resistance to a transgenic plant comprising pound which Specifically binds to one of Said the expression in particular cells, tissueS or organs of a plant, polypeptides or Said Second complex, and of a nucleic acid encoding an LRR receptor-like kinase, for 0126 d) identifying the compound. instance the LRR receptor-like kinase of the invention and defined above, wherein optionally Said nucleic acid is oper 0127. The invention also relates to a method for identi ably linked to a plant-operable promoter Sequence, for fying compounds or mixtures of compounds which specifi instance Said plant-operable promoter being the transcrip cally bind to a polypeptide of the invention, comprising: tional regulatory root promoter as defined earlier. 0128 a) combining a polypeptide of the invention 0116 Many pathogen resistance genes are LRR receptor with Said compound or mixtures of compounds like proteins, which initiate a signal transduction pathway in under conditions Suitable to allow complex forma the root cells, leading to the activation of defense mecha tion, and, nisms against the pathogen. The novel LRR receptor-like 0129 b) detecting complex formation, wherein the kinase might contribute to pathogen resistance to the trans presence of a complex identifies a molecule which genic plant when transformed herein. Specifically binds Said polypeptide. 0117 Yet another embodiment of the invention relates to methods to perform a functional analysis of the DNA 0.130. The invention further relates to the use of a mol Sequence according to the present invention, encoding a ecule identified by means of one of the methods described novel LRR receptor-like protein. For persons skilled in the above as a plant growth regulator or herbicide. art it is possible to make Single or double knock-out mutants 0131 The invention also relates to a method for the of Arabidopsis thaliana, wherein the DNA of the present production of a plant growth regulator or herbicide compo invention is inactivated. Also introduction of the antisense Sition comprising the Steps of one of the methods described sequence of the DNA of the invention, as well as the above and formulating the compounds obtained from Said overexpression of its Sense Sequence in a transgenic plant Steps in a Suitable form for the application in agriculture or can elucidate a specific feature in this transgenic plant. plant cell or tissue culture. Furthermore, the loss of function of this gene of the inven 0132) The invention also relates to the use of any of the tion by introduction of dominant negative mutations can nucleic acid molecules, vectors, polypeptides or antibodies result in interesting characteristics of the mutant plant. of described herein for modifying cell fate and/or plant 0118. Another aspect of the present invention is the development and/or plant morphology and/or plant bio revelation of a new target for herbicides or growth regula chemistry and/or plant physiology. tors. When Said gene of the invention is an essential gene for plant cell viability, it can be a target for a herbicide or growth 0133. The invention also relates to a diagnostic compo regulator. Sition comprising at least one of the nucleic acid molecules, vectors, polypeptides or antibodies of the invention. 0119) Therefore the invention relates to a method for identifying and obtaining proteins interacting with a (LRR 0.134. It is further important to notice that CLAVATA 1 receptor-like kinase) polypeptide of the invention compris has many analogues in other plant species, So it is likely that ing a Screening assay wherein Said polypeptide is used or also the promoter of RCH1, the RCH1 ORF or the complete expressed. RCH1 operon can be functional in those other plant species, 0120 In a preferred embodiment the invention relates to and possibly exert the same Specific activation pattern. This a method for identifying and obtaining proteins interacting can be investigated by root-specific reporter gene expression with a an LRR receptor-like kinase (protein) comprising a in rice mediated by the Arabidopsis thaliana RCH1 pro two-hybrid Screening System wherein a nucleic acid encod moter as described in example 5. ing a polypeptide of the invention as a bait and a cDNA 0135) Definitions and Elaborations to the Embodiments library as prey are expressed. 0136. The terms “protein(s)”, “peptide(s)" or “oligopep 0121 The invention also relates to a method for modu tide(s)”, when used herein refer to amino acids in a poly lating the interaction between an LRR receptor-like kinase meric form of any length. Said terms also include known (protein) of the invention and interacting proteins obtainable amino acid modifications Such as disulphide bond forma by a method as described above. tion, cysteinylation, oxidation, glutathionylation, methyla 0122) The invention further relates to a method for iden tion, acetylation, farnesylation, biotinylation, Stearoylation, tifying and obtaining compounds interacting with an LRR formylation, lipoic acid addition, phosphorylation, Sul receptor-like kinase (protein) comprising the steps of: phation, ubiquitination, myristoylation, palmitoylation, US 2004/OO67506 A1 Apr. 8, 2004 geranylgeranylation, cyclization (e.g. pyroglutamic acid for domain of a transcriptional activator as used in the yeast mation), oxidation, deamidation, dehydration, glycosylation two-hybrid System, phage coat proteins, (histidine)-tag, (e.g. pentoses, hexosamines, N-acetylhexosamines, deoxy glutathione S-transferase, protein A, maltose-binding pro hexoses, hexoses, Sialic acid etc.), acylation and radiolabels tein, dihydrofolate reductase, Tag 100 epitope (EETAR (e.g. 'I, I, S, 'C, P, PH) as well as non-naturally FQPGYRS), c-myc epitope (EQKLISEEDL), FLAG(R)- occurring amino acid residues, L-amino acid residues and epitope (DYKDDDK), lacZ, CMP (calmodulin-binding D-amino acid residues. peptide), HA epitope (YPYDVPDYA), protein C epitope 0137) “Homologues” or “Homologs” of a protein of the (EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK). invention are those peptides, oligopeptides, polypeptides, 0140 Deletional variants of a protein of the invention are proteins and enzymes which contain amino acid Substitu characterised by the removal of one or more amino acids tions, deletions and/or additions relative to the Said protein from the amino acid Sequence of Said protein. with respect to which they are a homolog, without altering 0141 Homologues of the peptides or polypeptides of the one or more of its functional properties, in particular without invention contain a number of amino acid Substitutions, reducing the activity of the resulting. For example, a deletions and/or additions relative to Said peptide or homolog of Said protein will consist of a bioactive amino polypepthide Such that Said homologues are still Substan acid Sequence variant of Said protein. To produce Such tially different from the peptides or polypeptides known in homologs, amino acids present in the Said protein can be the art. The difference between proteins can be calculated in replaced by other amino acids having Similar properties, for terms of% identidity according to methods well known in the example hydrophobicity, hydrophilicity, hydrophobic art. Preferably, the homologues of the peptides or polypep moment, antigenicity, propensity to form or break C-helical tides of the present invention comprise the amino acid Structures or B-sheet Structures, and So on. An Overview of sequence represented in SEQ ID NO 5, optionally compris physical and chemical properties of amino acids is given in ing minor amino acid Substitutions Such as those described Table 1. in table 1. Other homologues of the polypeptides of the invention have an amino acid Sequence which is at least 65% 0.138. Substitutional variants of a protein of the invention identical to the sequence represented in SEQID NO 4 or 19. are those in which at least one residue in Said protein amino acid Sequence has been removed and a different residue 0.142 Amino acid variants of a protein of the invention inserted in its place. Amino acid Substitutions ar typically of may readily be made using peptide Synthetic techniques well Single residues, but may be clustered depending upon func known in the art, Such as Solid phase peptide Synthesis and tional constraints placed upon the polypeptide; insertions the like, or by recombinant DNA manipulations. The will usually be of the order of about 1-10 amino acid manipulation of DNA sequences to produce variant proteins residues and deletions will range from about 1-20 residues. which manifest as Substitutional, insertional or deletional Preferably, amino acid Substitutions will comprise conser variants are well known in the art. For example, techniques Vative amino acid Substitutions, Such as those described for making Substitution mutations at predetermined Sites in Supra. DNA having known Sequence are well known to those skilled in the art, such as by M13 mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio), QuickChange TABLE 1. Site Directed mutagenesis kit (Stratagene, San Diego, Properties of naturally occurring amino acids: Calif.), PCR-mediated site-directed mutagenesis or other Charge properties/ Site-directed mutagenesis protocols. hydrophobicity Side group Amino Acid 0.143 “Derivatives” of a protein of the invention are those peptides, oligopeptides, polypeptides, proteins and nonpolar Aliphatic Ala Ile Lieu. hydrophobic Aliphatic Wall enzymes which comprise at least about five contiguous S- containing amino acid residues of Said polypeptide but which retain the Aromatic Met biological activity of Said protein and which may further Imino Phe, Trp comprise additional naturally-occurring, altered glycosy Pro polar uncharged Aliphatic Gly lated, acylated or non-naturally occurring amino acid resi Amide Asn Glin dues compared to the amino acid Sequence of a naturally Aromatic Tyr occurring form of Said polypeptide. Alternatively or in Hydroxyl Ser Thr Sulfhydryl Cys addition, a derivative may comprise one or more non-amino positively charged Basic Arg, His Lys acid Substituents compared to the amino acid Sequence of a negatively charged Acidic Asp, Gly naturally-occurring form of Said polypeptide, for example a reporter molecule or other ligand, covalently or non-co Valently bound to the amino acid Sequence Such as, for 0139 Insertional amino acid sequence variants of a pro example, a reporter molecule which is bound thereto to tein of the invention are those in which one or more amino facilitate its detection. Preferably, the derivates of the pro acid residues are introduced into a predetermined site in Said teins of the present invention comprise the amino acid acid protein. Insertions can comprise amino-terminal and/or car sequence as represented in SEQ ID NO 5. boxy-terminal fusions as well as intra-Sequence insertions of 0144. With “immunologically active' is meant that a Single or multiple amino acids. Generally, insertions within molecule or Specific fragments thereof Such as epitopes or the amino acid Sequence will be Smaller than amino or haptens are recognized by, i.e. bind to antibodies. Preferably, carboxyl terminal fusions, of the order of about 1 to 10 the immunologically active or fragments of the proteins of residues. Examples of amino- or carboxy-terminal fusion the present invention comprise the amino acid acid Sequence proteins or peptides include the binding domain or activation as represented in SEQ ID NO 5. US 2004/OO67506 A1 Apr. 8, 2004
0145 With “functional fragment” or “functional homo yribonucleotides or a combination of both, in a polymeric logue' is meant a protein which comprises at least about form of any length. Said terms furthermore include double 5,10, 20, 50, 75, 100, 200,300, 400, 500, 600, 700, 800, 900, stranded and single-stranded DNA and RNA. Said terms 1000, 1050, 1100, 1110, 1120, 1130, 1131, 1132, 1133 or also include known nucleotide modifications Such as methy 1134 contiguous amino acid residues of Said polypeptide but lation, cyclization and caps and Substitution of one or more which retains the biological activity of said protein. Prefer of the naturally occurring nucleotides with an analog Such as ably, the functional fragments or functional homologues of inosine. Modifications of nucleotides include the addition of the polypeptide or proteins of the invention comprise the acridine, amine, biotin, cascade blue, cholesterol, Cy3(E), amino acid sequence as represented in SEQ ID NO 5 but Cy5(R), Cy5.5(R) Dabcyl, digoxigenin, dinitrophenyl, Edans, wherein minor amino acid Substitutions are allowed (see 6-FAM, fluorescein, 3'-glyceryl, HEX, IRD-700, IRD-800, table 1). JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-SCR), SE, BODIPY(R), 0146 In the context of the current invention are embod Marina Blue(R), Pacific Blue(R), Oregon Green(E), Rhodamine ied homologs, derivatives and/or immunologically active Greene(E), Rhodamine RedE), Rhodol Green(E) and Texas fragments of any of the inventive LRR receptor-like kinase RedCR. Polynucleotide backbone modifications include protein or polypeptide or homolog, derivative or fragment methylphosphonate, 2'-OMe-methylphosphonate RNA, thereof as defined Supra. phosphorothiorate, RNA, 2'-OMeRNA. Base modifications 0147 “Antibodies' include monoclonal, polyclonal, syn include 2-amino-dA, 2-aminopurine, 3'-(ddA), thetic or heavy chain camel antibodies as well as fragments 3'dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N-Me of antibodies such as Fab, Fv or scFv fragments. Mono dA, abasic site (dSpacer), biotin dT, 2'-OMe-5Me-C, clonal antibodies can be prepared by the techniques as 2'-OMe-propynyl-C, 3'-(5-Me-dC), 3'-(ddC), 5-Br-dC, 5-1- described in e.g. Liddle and Cryer (1991) which comprise dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA, convert the fusion of mouse myeloma cells to Spleen cells derived ible dC, convertible dG, convertible dT, convertible dU, from immunized animals. Furthermore, antibodies or frag 7-deaza-dG, 8-Br-dG, 8-oxo-dG, O'-Me-dG, S6-DNP-dG, ments thereof to a molecule or fragments thereof can be 4-methyl-indole, 5-nitroindole, 2'-OMe-inosine, 2'-dl, obtained by using methods as described in e.g. Harlow and O'-phenyl-di, 4-methyl-indole, 2'-deoxynebularine, 5-ni Lane (1988). In the case of antibodies directed against Small troindole, 2-aminopurine, dP(purine analogue), dK(pyrimi peptides Such as fragments of a protein of the invention, Said dine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT, biotin peptides are generally coupled to a carrier protein before dT, carboxy-dT, O'-Me-dT, O'-triazol dT, 2'-OMe Immunization of animals. Such protein carriers include propynyl-U, 5-Br-dU,2'-dU, 5-F-du, 5-1-dU, O'-triazol dU keyhole limpet hemocyanin (KLH), bovine serum albumin and radiolabels (e.g. 'I, I, S, “C. P. P. H). Said (BSA), ovalbumin and Tetanus toxoid. The carrier protein terms also encompass peptide nucleic acids (PNAS), a DNA enhances the immune response of the animal and provides analogue in which the backbone is a pseudopeptide consist epitopes for T-cell receptor binding sites. The term “anti ing of N-(2-aminoethyl)-glycine units rather than a Sugar. bodies' furthermore includes derivatives thereof Such as PNAS mimic the behaviour of DNA and bind complemen labelled antibodies. Antibody labels include alkaline phos tary nucleic acid strands. The neutral backbone of PNA phatase, PKH2, PKH26, PKH67, fluorescein (FITC), Hoecst results in Stronger binding and greater Specificity than nor 33258, R-phycoerythrin (PE), rhodamine (TRITC), Quan mally achieved. In addition, the unique chemical, physical tum Red, Texas Red, Cy3, biotin, agarose, peroxidase, gold and biological properties of PNA have been exploited to spheres and radiolabels (e.g. 'I, I, S, “C. P. P. produce powerful biomolecular tools, antisense and antigene H). Tools in molecular biology relying on antibodies agents, molecular probes and biosensors. With "recombinant against a protein include protein gel blot analysis, Screening DNA molecule” or “chimeric gene” is meant a hybrid DNA of expression libraries allowing gene identification, protein produced by joining pieces of DNA from different sources. quantitative methods including ELISA and RIA, immunoaf With “heterologous nucleotide sequence” is intended a finity purification of proteins, immunoprecipitation of pro Sequence that is not naturally occurring with the promoter teins (e.g. Magyar et al. 1997) and immunolocalization. Sequence. While this nucleotide Sequence is heterologous to Other uses of antibodies and especially of peptide antibodies the promoter Sequence, it may be homologous, or native, or include the study of proteolytic processing (Loffler et al. heterologous, or foreign, to the plant host. “Sense Strand” 1994, Woulfe et al. 1994), determination of protein active refers to the strand of a double-stranded DNA molecule that Sites (Lerner 1982), the study of precursor and post-trans is homologous to a mRNA transcript thereof. The “anti lational processing (Baron and Baltimore 1982, Lerner et al. Sense Strand” contains an inverted Sequence which is 1981, Semler et al. 1982), identification of protein domains complementary to that of the “Sense Strand'. involved in protein-protein interactions (Murakami et al. 0150. A “coding sequence” or “open reading frame” or 1992) and the study of exon usage in gene expression “ORF is defined as a nucleotide sequence that can be (Tamura et al. 1991). transcribed into mRNA and/or translated into a polypeptide 0148 Embodied in the current invention are antibodies when placed under the control of appropriate regulatory recognizing a LRR receptor-like kinase polypeptide or pro Sequences, i.e. when Said coding Sequence or ORF is present tein, or homolog, derivative or fragment thereof as defined in an expressible format. Said coding Sequence of ORF is Supra or Specific epitopes of Said polypeptide or protein. bounded by a 5' translation start codon and a 3’ translation Stop codon. A coding Sequence or ORF can include, but is 014.9 The terms “gene(s)”, “polynucleotide(s)”, “nucleic not limited to RNA, mRNA, cDNA, recombinant nucleotide acid sequence(s)”, “nucleotide sequence(s)”, “DNA Sequences, Synthetically manufactured nucleotide Sequences Sequence(s) or “nucleic acid molecule(s)”, when used or genomic DNA. Said coding sequence or ORF can be herein refer to nucleotides, either ribonucleotides or deox interrupted by intervening nucleic acid Sequences. US 2004/OO67506 A1 Apr. 8, 2004 12
hybridization proceSS can also occur with one of the comple TABLE 2 mentary nucleic acids immobilized to a matrix Such as magnetic beads, Sepharose beads or any other resin. Tools in Degeneracy of the genetic code. molecular biology relying on Such a process include the Three- One isolation of poly (A+) mRNA. The hybridization process can letter letter furthermore occur with one of the complementary nucleic Amino Acid code code Possible codons acids immobilized to a Solid Support Such as a nitrocellulose Alanine Ala A. GCA GCC GCG GCU or nylon membrane or immobilized by e.g. photolitography Arginine Arg R AGA AGG CGA CGC CGG CGU Asparagine Asn N AAC AAU to e.g. a silicious glass Support (the latter known as nucleic Aspartic Acid Asp D GAC GAU acid arrays or microarrays or as nucleic acid chips). Tools in Cysteine Cys C UGC UGU molecular biology relying on Such a proceSS include RNA Glutamic Acid Glu E GAA GAG and DNA gel blot analysis, colony hybridization, plaque Glutamine Glin Q CAA. CAG hybridization, in situ hybridization and microarray hybrid Glycine Gly G GGA. GGC GGG GGU Histidine His H CAC CAU ization. In order to allow hybridization to occur, the nucleic Isoleucine Ile I AUA AUG AUU acid molecules are generally thermally or chemically dena Leucine Telu L UUA UUG CUA CUC CUG CUU tured to melt a double Strand into two Single Strands and/or Lysine Lys K AAA AAG Methionine Met M AUG to remove hairpins or other Secondary Structures from Single Phenylalanine Phe F UUC UUU Stranded nucleic acids. The Stringency of hybridization is Proline Pro P CCA CCC CCG CCU influenced by conditions Such as temperature, Salt concen Serine Ser S AGC AGU UCA, UCA, UCG UCU tration and hybridization buffer composition. High strin Threonine Thr T ACA ACC ACG ACU Tryptophan Trp W UGG gency conditions for hybridization include high temperature Tyrosine Tyr Y UAC UAU and/or low Salt concentration (salts include NaCl and Na Waline Wall W GUA.. GUC GUG GUU citrate) and/or the inclusion of formamide in the hybridiza Possible 'STOP' codons tion buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridization buffer and/or UAA, UAG UGA exclusion of compounds Such as dextran Sulfate or polyeth ylene glycol (promoting molecular crowding) from the 0151 Genes and coding sequences essentially encoding hybridization buffer. Conventional hybridization conditions the same protein but isolated from different Sources can are described in e.g. Sambrook et al. (1989) but the skilled consist of Substantially divergent nucleic acid sequences. craftsman will appreciate that numerous different hybridiza Reciprocally, Substantially divergent nucleic acid Sequences tion conditions can be designed in function of the known or can be designed to effect expression of essentially the same the expected homology and/or length of the nucleic acid protein. Said nucleic acid Sequences are the result of e.g. the Sequence. Sufficiently low Stringency hybridization condi existence of different alleles of a given gene, of the degen tions are particularly preferred to isolate nucleic acids het eracy of the genetic code or of differences in codon usage. erologous to the DNA sequences of the invention defined Thus, as indicated in Table 2, amino acids Such as methion Supra. Elements contributing to Said heterology include ine and tryptophan ar encoded by a single codon whereas allelism, degeneration of the genetic code and differences in other amino acids Such as arginine, leucine and Serine can preferred codon usage as discussed Supra. each be translated from up to six different codons. Differ 0153 Clearly, the current invention embodies the use of ences in preferred codon usage are illustrated below for the inventive DNA sequences encoding a LRR receptor-like Agrobacterium tumefaciens (a bacterium), A. thaliana, M. kinase, homolog, derivative and/or immunologically frag Sativa (two dicotyledonous plants) and Oryza Sativa (a ment thereof as defined higher in any method of hybridiza monocotyledonous plant). These examples were extracted tion. The current invention furthermore also relates to DNA from (http://www.kazusa.or.jp/codon). To give one example, Sequences Specifically hybridizing to Said inventive DNA the codon GGC (for glycine) is the most frequently used Sequences. codon in A. tumefaciens (36.2% ), is the second most frequently used codon in O. Sativa but is used at much lower 0154 DNA sequences as defined in the current invention frequencies in A. thaliana and M. Saiiva (9% and 8.4%, can be interrupted by intervening Sequences. With "inter respectively). Of the four possible codons encoding glycine vening Sequences” is meant any nucleic acid Sequence (see Table 6), said GGC codon is most preferably used in A. which disrupts a coding Sequence comprising Said inventive tumefaciens and O. Sativa. However, in A. thaliana this is DNA sequence or which disrupts the expressible format of the GGA (and GGU) codon whereas in M. Sativa this is the a DNA sequence comprising Said inventive DNA sequence. GGU (and GGA) codon. Removal of the intervening Sequence restores Said coding Sequence or Said expressible format. Examples of interven 0152 “Hybridization” is the process wherein substan ing Sequences include introns, mobilizable DNA sequences tially homologous complementary nucleotide Sequences Such as transposons and DNA tags Such as e.g. a T-DNA. anneal to each other. The hybridization process can occur With “mobilizable DNA sequence” is meant any DNA entirely is in Solution, I.e. both complementary nucleic acids Sequence that can be mobilized as the result of a recombi are in Solution. Tools in molecular biology relying on Such nation event. a process include the polymerase chain reaction (PCR; and all methods based thereon), Subtractive hybridization, ran O155 To effect expression of a protein in a cell, tissue or dom primer eXtension, nuclease S1 mapping, primer exten organ, preferably of plant origin, either the protein may be Sion, reverse transcription, cDNA synthesis, differential dis introduced directly to Said cell, Such as by microinjection or play of RNAS, and DNA sequence determination. The ballistic means or alternatively, an isolated nucleic acid US 2004/OO67506 A1 Apr. 8, 2004 molecule encoding Said protein may be introduced into Said t-butyl (t-Bu), anisyl, benzyl (Bzl), trifluroacetyl (Tfa), cell, tissue or organ in an expressible format. N-hydroxysuccinimide (ONSu, OSu), benzoyl (BZ), 4-me thylbenzyl (Meb), thioanizyl, thiocresyl, benzyloxymethyl 0156 Preferably, the DNA sequence of the invention (Bom), 4-nitrophenyl (ONp), benzyloxycarbonyl (Z), 2-ni comprises a coding sequence or open reading frame (ORF) trobenzoyl (NBz), 2-nitrophenylsulphenyl (Nps), 4-toluene encoding a LRR receptor-like kinase or a homolog or sulphonyl (Tosyl, Tos), pentafluorophenyl (Pfp), diphenyl derivative thereof or an immunologically active fragment methyl (Dpm), 2-chlorobenzyloxycarbonyl (CI-Z), 2,4,5- thereof as defined Supra. The preferred protein of the inven trichlorophenyl, 2-bromobenzyloxycarbonyl (Br-Z), tion comprises the amino acid Sequence of Said LRR recep tripheylmethyl (Trityl, Trt), and 2,5,7,8-pentamethylchro tor-like kinase. man-6-sulphonyl (Pmc). During chain assembly, Fmoc or 0157 With “vector” or “vector sequence” is meant a Boc are removed resulting in an activated amino-terminus of DNA sequence which can be introduced in an organism by the amino acid residue bound to the growing chain. The transformation and can be stably maintained in Said organ carboxy-terminus of the incoming amino acid is activated by ism. Vector maintenance is possible in e.g. cultures of conversion into a highly reactive ester, e.g. by HBTU. With Escherichia coli, A. tumefaciens, Saccharomyces cerevisiae current technologies (e.g. PerSeptive Biosystems 9050 syn or Schizosaccharomyces pombe. Other vectors such as thesizer, Applied Biosystems Model 431 A Peptide Synthe phagemids and coSmid vectors can be maintained and mul sizer), linear peptides of up to 50 residues can be manufac tiplied in bacteria and/or viruses. Vector Sequences generally tured. A number of guidelines is available to produce comprise a set of unique sites recognized by restriction peptides that are Suitable for use in biological Systems enzymes, the multiple cloning site (MCS), wherein one or including (i) limiting the use of difficult amino acids Such as more non-vector Sequence(s) can be inserted. cys, met, trp (easily oxidized and/or degraded during peptide Synthesis) or arg; (ii) minimize hydrophobic amino acids 0158 With “non-vector sequence” is accordingly meant (can impair peptide Solubility); and (iii) prevent an amino a DNA sequence which is integrated in one or more of the terminal glutamic acid (can cyclize to pyroglutamate). sites of the MCS comprised within a vector. 0162 By “expressible format' is meant that the isolated 0159) “Expression vectors” form a subset of vectors nucleic acid molecule is in a form Suitable for being tran which, by virtue of comprising the appropriate regulatory Scribed into mRNA and/or translated to produce a protein, Sequences enabling the creation of an expressible format for either constitutively or following induction by an intracel the inserted non-vector Sequence(s), thus allowing expres lular or extracellular signal, Such as an environmental Stimu Sion of the protein encoded by said non-vector sequence(s). lus or StreSS (mitogens, anoxia, hypoxia, temperature, Salt, Expression vectors are known in the art enabling protein light, dehydration, etc) or a chemical compound Such as expression in organisms including bacteria (e.g. E. coli), IPTG (isopropyl-f-D-thiogalactopyranoside) or Such as an fungi (e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect antibiotic (tetracycline, amplicillin, rifampicin, kanamycin), cells (e.g. baculoviral expression vectors), animal cells (e.g. hormone (e.g. gibberellin, auxin, cytokinin, glucocorticoid, COS or CHO cells) and plant cells (e.g. potato virusX-based brassinosteroid, ethylene, abscisic acid etc), hormone ana expression vectors, See e.g. Vance et al. 1998 logue (iodoacetic acid (IAA), 2,4-D, etc), metal (zinc, copper, WO984.4097). iron, etc), or dexamethasone, amongst others. AS will be 0160 The current invention clearly includes any vector known to those skilled in the art, expression of a functional or expression vector comprising a non-vector DNA protein may also require one or more post-translational Sequence comprising the promoter Sequence according to modifications, Such as glycosylation, phosphorylation, the present invention or a non-vector Sequence encoding a dephosphorylation, or one or more protein-protein interac LRR receptor-like kinase, homolog, derivative and/or tions, amongst others. All Such processes are included within immunologically active fragment thereof as defined Supra. the scope of the term “expressible format”. 0163 Preferably, expression of a protein in a specific cell, 0.161. As an alternative to expression vector-mediated protein production in biological Systems, chemical protein tissue, or organ, preferably of plant origin, is effected by Synthesis can be applied. Synthetic peptides can be manu introducing and expressing an isolated nucleic acid molecule factured in Solution phase or in Solid phase. Solid phase encoding Said protein, Such as a cDNA molecule, genomic peptide synthesis (Merrifield 1963) is, however, the most gene, Synthetic oligonucleotide molecule, mRNA molecule common way and involves the Sequential addition of amino or open reading frame, to Said cell, tissue or organ, wherein acids to create a linear peptide chain. Solid phase peptide Said nucleic acid molecule is placed operably in connection Synthesis includes cycles consisting of three steps: (i) immo with Suitable regulatory Sequences including a promoter, bilization of the carboxy-terminal amino acid of the growing preferably a plant-expressible promoter, and a terminator peptide chain to a Solid Support or resin; (ii) chain assembly, Sequence. a process consisting of activation, coupling and deprotection 0164) “Regulatory sequence” refers to control DNA of the amino acid to be added to the growing peptide chain; Sequences which are necessary to affect the expression of and (iii) cleavage involving removal of the completed pep coding Sequences to which they are ligated. The nature of tide chain from the resin and removal of the protecting Such control Sequences differS depending upon the host groups from the amino acid Side chains. Common organism. In prokaryotes, control Sequences generally approaches in Solid phase peptide Synthesis include Fmoc/ include promoters, ribosomal binding Sites, and terminators. tlu (9-fluorenylmethyloxycarbonyl/t-butyl) and Boc (t-bu In eukaryotes generally control Sequences include promot tyloxycarbonyl) as the amino-terminal protecting groups of ers, terminators and enhancers or Silencers. The term “con amino acids. Amino acid Side chain protecting groups trol Sequence' is intended to include, at a minimum, all include methyl (Me), formyl (CHO), ethyl (Et), acetyl (Ac), components the presence of which are necessary for expres US 2004/OO67506 A1 Apr. 8, 2004
Sion, and may also include additional advantageous compo the plant or alternatively, throughout the plant under a nents and which determines when, how much and where a Specific Set of conditions, Such as following induction of Specific gene is expressed. gene expression by a chemical compound or other elicitor. 0.165 Reference herein to a “promoter” is to be taken in 0172 Preferably, the regulatable promoter used in the its broadest context and includes the transcriptional regula performance of the present invention conferS expression in tory Sequences derived from a classical eukaryotic genomic a specific location within the plant, either constitutively or gene, including the TATA box which is required for accurate following induction, however not in the whole plant under transcription initiation, with or without a CCAAT box any circumstances. Included within the Scope of Such pro Sequence and additional regulatory elements (i.e. upstream moters are cell-Specific promoter Sequences, tissue-specific activating Sequences, enhancers and Silencers) which alter promoter Sequences, organ-Specific promoter Sequences, gene expression in response to developmental and/or exter cell cycle Specific gene promoter Sequences, inducible pro nal Stimuli, or in a tissue-specific manner. moter Sequences and constitutive promoter Sequences that 0166 The term “promoter” also includes the transcrip have been modified to confer expression in a particular part tional regulatory Sequences of a classical prokaryotic gene, of the plant at any one time, Such as by integration of Said in which case it may include a -35 box sequence and/or a constitutive promoter within a transposable genetic element -10 box transcriptional regulatory Sequences. (Ac, DS, Spm, En, or other transposon). The skilled crafts man will understand that a “constitutive promoter' is a 0167 The term “promoter' is also used to describe a promoter that is transcriptionally active in an organism, Synthetic or fusion molecule, or derivative which confers, preferably a plant, during most, but not necessarily all activates or enhances expression of a nucleic acid molecule phases of its growth and development. Similarly, the skilled in a cell, tissue or organ. craftsman will understand that a "ubiquitous promoter' is a 0168 The term “functional part” when referred to nucleic promoter that is transcriptionally active throughout most, acids in relation to the above defined transcriptional regu but not necessarily all parts of an organism, preferably a latory Sequences, refers to a part or parts of the nucleic acid plant. having the activity to Specifically drive or promote tran 0173 Generally by “weak promoter” is intended a pro Scription from Sequences which are located downstream of moter that drives expression of a coding Sequence at a low Said nucleic acid Sequence. level. By “low level” is intended at levels of about /10,000 0169 Promoters may contain additional copies of one or transcripts to about /100,000 transcripts, to about /500.0000 more specific regulatory elements, to further enhance transcripts. Conversely, a “strong promoter' drives expres expression and/or to alter the Spatial expression and/or Sion of a coding Sequence at high level, or at about /10 temporal expression of a nucleic acid molecule to which it transcripts to about /100 transcripts to about/1,000 transcripts. is operably connected. Such regulatory elements may be placed adjacent to a heterologous promoter Sequence to 0.174. The term “cell-specific' shall be taken to indicate drive expression of a nucleic acid molecule in response to that expression is predominantly in a particular cell or e.g. copper, glucocorticoids, dexamethasone, tetracycline, cell-type, preferably of plant origin, albeit not necessarily gibberellin, cAMP, abscisic acid, auxin, wounding, ethylene, exclusively in Said cell or cell-type. jasmonate or Salicylic acid or to confer expression of a 0.175 Similarly, the term “tissue-specific' shall be taken nucleic acid molecule to Specific cells, tissueS or organs Such to indicate that expression is predominantly in a particular as meristems, leaves, roots, embryo, flowers, Seeds or fruits. tissue or tissue-type, preferably of plant origin, albeit not 0170 In the context of the present invention, the pro necessarily exclusively in Said tissue or tissue-type. moter preferably is a plant-expressible promoter Sequence. 0176) Similarly, the term “organ-specific' shall be taken Promoters, however, that also function or solely function in to indicate that expression is predominantly in a particular non-plant cells Such as bacteria, yeast cells, insect cells and organ, preferably of plant origin, albeit not necessarily animal cells are not excluded from the invention. By “plant expressible' is meant that the promoter Sequence, including exclusively in Said organ. "Root Specific' means that the any additional regulatory elements added thereto or con promoter is expressed in the root only and not in other tained therein, is at least capable of inducing, conferring, tissueS of the plant. activating or enhancing expression in a plant cell, tissue or 0177. By “root-preferred” it is intended that expression of organ, preferably a monocotyledonous or dicotyledonous the heterologous nucleotide Sequence is most abundant root, plant cell, tissue, or organ. The terms “plant-operable” and but could also have low expression levels elsewhere in the “operable in a plant' when used herein, in respect of a plant. While some level of expression of the heterologous promoter Sequence, Shall be taken to be equivalent to a nucleotide Sequence occurs in other plant tissue types, plant-expressible promoter Sequence. expression occurs most abundantly in the root including 0171 In the present context, a “regulatable promoter primary, lateral and adventitious roots. Sequence' is a promoter that is capable of conferring expres 0.178 By “root” is intended any part of the root structure, Sion on a structural gene in a particular cell, tissue, or organ including, but not limited to, the root cap, apical meristem, or group of cells, tissueS or organs of a plant, optionally protoderm, ground meristem, procambium, endodermis, under Specific conditions, however does generally not confer cortex, vascular cortex, epidermis, and the like. expression throughout the plant under all conditions. Accordingly, a regulatable promoter Sequence may be a 0179 Similarly, the term “cell cycle specific” shall be promoter Sequence that conferS expression on a gene to taken to indicate that expression is predominantly cyclic and which it is operably connected in a particular location within occurring in one or more, not necessarily consecutive phases US 2004/OO67506 A1 Apr. 8, 2004 15 of the cell cycle albeit not necessarily exclusively in cycling in Such a way that expression of the coding Sequence is cells, preferably of plant origin. achieved under conditions compatible with the control Sequences. In case the control Sequence is a promoter, R is 0180 Contrarily, the term ubiquitous shall be taken to obvious for a skilled person that double-stranded nucleic indicate that expression is throughout the entire oranism. acid is preferably used. 0181 Those skilled in the art will be aware that an 0186 Examples of promoters suitable for use in gene “inducible promoter' is a promoter the transcriptional activ constructs of the present invention include those listed in ity of which is increased or induced in response to a Table 3, amongst others. The promoters listed in Table 5 are developmental, chemical, environmental, or physical Stimu provided for the purposes of exemplification only and the lus. Similarly, the skilled craftsman will understand that a “constitutive promoter' is a promoter that is transcription present invention is not to be limited by the list provided ally active throughout most, but not necessarily all parts of therein. Those skilled in the art will readily be in a position an organism, preferably a plant, during most, but not nec to provide additional promoters that are useful in performing essarily all phases of its growth and development. the present invention. 0187. In the case of constitutive promoters or promoters 0182 Those skilled in the art will readily be capable of that induce expression throughout the entire plant, it is Selecting appropriate promoter Sequences for use in regu preferred that Such Sequences are modified by the addition of lating appropriate expression of the LRR receptor-like nucleotide Sequences derived from one or more of the kinase as described Supra from publicly-available or readily tissue-specific promoters listed in Table 8, or alternatively, available Sources, without undue experimentation. nucleotide Sequences derived from one or more of the 0183 Placing a nucleic acid molecule under the regula above-mentioned tissue-specific inducible promoters, to tory control of a promoter Sequence, or in operable connec confer tissue-specificity thereon. For example, the CaMV tion with a promoter Sequence, means positioning Said 35S promoter may be modified by the addition of maize nucleic acid molecule Such that expression is controlled by Adh1 promoter Sequence, to confer anaerobically-regulated the promoter Sequence. A promoter is usually, but not root-specific expression thereon, as described previously necessarily, positioned upstream, or at the 5'-end, and within (Ellis et al., 1987). Another example describes conferring 2 kb of the Start Site of transcription, of the nucleic acid root Specific or root abundant gene expression by fusing the molecule which it regulates. In the construction of heterolo CaMV35S promoter to elements of the maize glycine-rich gous promoter/Structural gene combinations it is generally protein GRP3 gene (Feix and Wulff 2000–WO0015662). preferred to position the promoter at a distance from the Such modifications can be achieved by routine experimen gene transcription Start Site that is approximately the same as tation by those skilled in the art. the distance between that promoter and the gene it controls 0188 The term “terminator" refers to a DNA sequence at in its natural Setting (i.e., the gene from which the promoter the end of a transcriptional unit which Signals termination of is derived). AS is known in the art, Some variation in this transcription. Terminators are 3'-non-translated DNA distance can be accommodated without loSS of promoter Sequences containing a polyadenylation Signal, which facili function. Similarly, the preferred positioning of a regulatory tates the addition of polyadenylate Sequences to the 3'-end of Sequence element with respect to a heterologous gene to be a primary transcript. Terminators active in cells derived from placed under its control is defined by the positioning of the Viruses, yeasts, moulds, bacteria, insects, birds, mammals element in its natural Setting (i.e., the gene from which it is and plants are known and described in the literature. They derived). Again, as is known in the art, Some variation in this may be isolated from bacteria, fungi, Viruses, animals and/or distance can also occur. plants. 0184) “Expression” means the production of a protein or 0189 Examples of terminators particularly suitable for nucleotide Sequence in the cell itself or in a cell-free System. use in the gene constructs of the present invention include It includes transcription into an RNA product, post-tran the Agrobacterium tumefaciens nopaline synthase (NOS) Scriptional modification and/or translation to a protein prod gene terminator, the Agrobacterium tumefaciens octopine uct or polypeptide from a DNA encoding that product, as Synthase (OCS) gene terminator Sequence, the Cauliflower well as possible post-translational modifications. mosaic virus (CaMV) 35S gene terminator sequence, the 0185 “Operably linked” refers to a juxtaposition wherein Oryza Sativa ADP-glucose pyrophosphorylase terminator the components So described are in a relationship permitting Sequence (t3'Bt2), the Zea mayS Zein gene terminator them to function in their intended manner. A control Sequence, the rbcS-1A gene terminator, and the rbcS-3A gene Sequence “operably linked' to a coding Sequence is ligated terminator Sequences, amongst others.
TABLE 3 Exemplary plant-expressible promoters for use in the performance of the present invention
EXPRESSION GENE SOURCE PATTERN REFERENCE
I: CELL-SPECIFIC, TISSUE-SPECIFIC, AND ORGAN-SPECIFIC PROMOTERS C-amylase (Amy32b) Aleurone Lanahan et al, Plant Cell 4: 203-211, 1992: Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin ?-like Aleurone Cejudo et al, Plant Mol Biol 20: 849–856, gene 1992 US 2004/OO67506 A1 Apr. 8, 2004 16
TABLE 3-continued Exemplary plant-expressible promoters for use in the performance of the present invention Agrobacterium Cambium Nilsson et al., Physiol Plant 100: 456-462, rhizogenes rollB 1997 AtPRP4 Flowers http://salus.medium.edu/mmg/tierneV/html chalcone synthase Flowers Van der Meer et al, Plant Mol Biol 15: 95-109, 1990 (chSA) LATS2 Anther Twell et al, Mol Gen Genet 217: 240-245, 1989 apetala-3 Flowers chitinase fruit (berries, grapes, Thomas et al. CSIRO Plant Industry, etc) Urrbrae, South Australia, Australia; http://winetitles.com.au/gwrdcfcsh95-1.html rbcs-3A green tissue (eg leaf) Lam et al, Plant Cell 2: 857-866, 1990; Tucker et al., Plant Physiol 113: 1303–1308, 1992 leaf-specific genes Lea Baszczynski et al, Nucl Acid Res 16: 4732, 1988 AtPRP4 Lea http://salus.medium.edu/mmg/tierney/html chlorella virus Lea Mitra and Higgins, Plant Mol Biol 26: 85–93, 1994 adenine methyltransferase gene promoter aldP gene promoter Lea Kagaya et al, Mol Gen. Genet 248: 668-674, from rice 1995 rbcs promoter from Lea Kyozuka et al, Plant Physiol 102:991-1000, rice or tomato 1993 Pinus cab-6 Lea Yamamoto et al, Plant Cell Physiol 35: 773–778, 1994 rubisco promoter Lea cab (chlorophyll Lea afb?binding protein pea Blec4 gene Vegetative and floral Mandaci and Dobres, Plant Mol Biol epidermal tissues 34:961-965 SAM22 senescent leaf Crowell et al, Plant Mol Biol 18:459–466, 1992 ltp gene (lipid Fleming et al, Plant J 2: 855–862, 1992 transfer gene) R. japonicum nif nodule U.S. Pat. No. 48O31.65 gene B. japonicum nifH nodule U.S. Pat. No. SOO819.4 gene GmNOD40 nodule Yang et al, Plant J 3: 573-585, 1993 PEP carboxylase nodule Pathirana et al, Plant Mol Biol 20: 437-450, (PEPC) 1992 Leghaemoglobin nodule Gordon et al, J Exp Bot 44: 1453-1465, (Lb) 1993 Tungro baciliiform phloem Bhattacharyya-Pakrasi et al, Plant J 4: 71-79, virus gene 1992 pollen-specific genes pollen; microspore Albani et al, Plant Mol Biol 15: 605, 1990; Albani et al, Plant Mol Biol 16:501, 1991 Zim13 pollen Guerrero et al, Mol Gen. Genet 224: 161-168, 1993 apg gene microspore Twell et al, Sex Plant Reprod 6:217-224, 1993 maize pollen-specific pollen Hamilton et al., Plant Mol Biol 18: 211-218, gene 1992 Sunflower pollen pollen Baltz et al, Plant J 2: 713–721, 1992 expressed gene B. napus pollen pollen:anther; tapetum Arnoldo et al., J. Cell Bioch m, Abstract No. specific gene Y101, 204, 1992 root-expressible OOS Tingey et al, EMBO J 6: 1, 1987 genes Tobacco auxin root tip Van der Zaal et al, Plant Mol Biol 16:983, inducible gene 1991 B-tubulin OO Oppenheimer et al, Gene 63: 87, 1988 Tobacco root OO Conkling et al, Plant Physiol 93: 1203, 1990 specific genes B. napus G1-3b OO U.S. Pat. No. 54O1836 gene SbFRP1 OOS Suzuki et al, Plant Mol Biol 21: 109-119, 1993 US 2004/OO67506 A1 Apr. 8, 2004 17
TABLE 3-continued Exemplary plant-expressible promoters for use in the performance of the present invention AtPRP1; AtPRP3 roots; root hairs http://salus.medium.edu/mmg/tierney/html RD2 gene rOOt cortex http://www2.cnsu.edu/ncSufresearch TobRB7 gene root vasculature http://www2.cnsu.edu/ncSufresearch AtPRP4 leaves; flowers; lateral http://salus.medium.edu/mmg/tierney/html root primordia seed-specific genes SeeC Simon et al, Plant Mol Biol 5: 191, 1985; Scofield et al, J Biol Chem 262: 12202, 1987: Baszczynski et al, Plant Mol Biol 14: 633, 1990 Brazil Nut albumin SeeC Pearson et al, Plant Mol Biol 18: 235-245, 1992 Legumin SeeC Ellis et al, Plant Mol Biol 10:203-214, 1988 Glutelin (rice) SeeC Takaiwa et al, Mol Gen. Genet 208: 15-22, 1986; Takaiwa et al, FEBS Lett 221: 43–47, 1987 Zein SeeC Matzke et al, Plant Mol Biol 14:323-32 1990 Nap A SeeC Stalberg et al, Planta 199: 515-519, 1996 wheat LMW and endosperm Mol Gen Genet 216: 81–90, 1989; Nucl HMW glutenin-1 Acids Res 17: 461–462, 1989 wheat SPA SeeC Albani et al, Plant Cell 9: 171-184, 1997 cZ19B1, maize 19 SeeC WOOO11177 kDa zein millps, maiz SeeC WOOO11177 myoinositol-1-Pi synthase wheat Cl, f, Y-gliadins CCOSC EMBO J 3: 1409-1415, 1984 barley ltr1 promoter CCOSC barley B1, C, D, CCOSC Theor Appl Gen98: 1253–1262, 1999; Plant hordein J 4:343–355, 1993; Mol Gen Genet 250: 750-60, 1996 barley DOF CCOSC Mena et al, Plant J 116:53–62, 1998 blz2 CCOSC EP991O6056.7 synthetic promoter eCOSel Vicente-Carbajosa et al, Plant J 13: 629–640, 1998 rice prolamin NRP33 CCOSC Wu et al, Plant Cell Physiol 39: 885–889, 1998 rice C-globulin Glb-1 CCOSC Wu et al, Plant Cell Physiol 39: 885–889, 1998 maize END genes CCOSC WOOO12733 barley END1 CCOSC barley NUC1 nucellus WO98O8961 rice OSH1 embryo Sato et al., Proc Natl Acad Sci USA 93:8117-8122, 1996 rice C-globulin CCOSC Nakase et al, Plant Mol Biol 33: 513–522, REB/OHP-1 1997 rice ADP-glucose PP CCOSC Trans Res 6: 157-168, 1997 maize ESR gene CCOSC Plant J 12: 235-246, 1997 family sorgum Y-kafirin CCOSC Plant Mol Biol 32: 1029-1035, 1996 KNOX embryo Postma-Haarsma et al, Plant Mol Biol 39: 257-271, 1999 rice oleosin embryo and aleuron Wu et al, J Biochem 123: 386, 1998 Sunflower oleosin seed (embryo and dry Cummins et al, Plant Mol Biol 19: 873-876, seed) 1992 LEAFY shoot meristem Weigel et al, Cell 69: 843–859, 1992 Arabidopsis thaliana shoot meristem Accession number AJ131822 knat1 Maius domestica shoot meristem Accession number Z71981 kn1 CLAVATA1 shoot meristem Accession number AFO49870 stigma-specific stigma Nasrallah et al, Proc Natl Acad Sci USA genes 85:5551, 1988; Tricket al, Plant Mol Biol 15: 203, 1990 class I patatin gene tuber Liu et al, Plant Mol Biol 153: 386–395, 1991 PCNA rice meristem Kosugi et al, Nucl Acids Res 19: 1571-1576, 1991; Kosugi and Ohashi, Plant Cell 9: 1607-1619, 1997 Pea TubA1 tubulin Dividing cells Stotz and Long, Plant Mol Biol 41: 601-614, 1999 Arabidopsis cdc2a cycling cells Chung and Parish, FEBS Let 362: 215–219, 1995 Arabidopsis Rop1A Anthers; mature pollen + pollen Li et al, Plant Physiol 118: 407-417, 1998 tubes Arabidopsis AtDMC1 Meiosis-associated Klimyuk and Jones, Plant J 11:1-14, 1997 US 2004/OO67506 A1 Apr. 8, 2004 18
TABLE 3-continued Exemplary plant-expressible promoters for use in the performance of the present invention Pea PS-IAA4/5 and Auxin-inducible Wong et al, Plant J 9: 587-599, 1996 PS-AA6 Pea Meristematic tissues; Zhou et al, Plant J 12:921-930, 1997 famesyltransferase phloem near growing tissues; light-and sugar-repressed Tobacco (N. Dividing cells/ Trehin et al, Plant Mol. Biol. 35: 667-672, Sylvestris) cyclin meristematic tissue 1997 B1:1 Catharanthus roseus Dividing cells/ Ito et al, Plant J 11:983–992, 1997 Mitotic cyclins CYS meristematic tissue (A-type) and CYM (B-type) Arabidopsis cyc1At Dividing cells/ Shaul et al, Proc Natl Acad Sci USA (=cyc B1;1) and meristematic tissue 93: 4868–4872, 1996 cyc3aAt (A-type) Arabidopsis tef1 Dividing cells/ Regadet al, Mol Gen. Genet 248: 703-711, promoter box meristematic tissue 1995 Catharanthus roseus Dividing cells/ Ito et al, Plant Mol Biol 24: 863–878, 1994 cycO7 meristematic tissue II: EXEMPLARY CONSTITUTIVE PROMOTERS Actin constitutive McElroy et al, Plant Cell 2:163–171, 1990 CAMV 35S constitutive Odell et al, Nature 313: 810-812, 1985 CaMV 19S constitutive Nilsson et al., Physiol Plant 100: 456-462, 1997 GOS2 constitutive de Pater et al, Plant J 2: 837-844, 1992 Ubiquitin constitutive Christensen et al, Plant Mol Biol 18: 675-689, 1992 rice cyclophilin constitutive Buchholz et al, Plant Mol Biol 25: 837-843, 1994 maize histone H3 constitutive Lepetit et al, Mol Gen. Genet 231: 276-285, 1992 alfalfa histone H3 constitutive Wu et al, Nucleic Acids Res 17: 3057-3063, 1989; Wu et al, Plant Mol Biol 11: 641–649, 1988 actin 2 constitutive An et al, Plant J 10: 107-121, 1996
III: EXEMPLARY STRESS-INDUCIBLE PROMOTERS
NAME STRESS REFERENCE P5CS (delta(1)- salt, water Zhang et al, Plant Sci 129: 81–89, 1997 pyrroline-5- carboxylate syntase) cor15a cold Hajela et al, Plant Physiol 93: 1246-1252, 1990 cor15b cold Wlihelmet al, Plant Mol Biol 23: 1073-1077, 1993 cor15a (-305 to +78 cold, drought Baker et al, Plant Mol Biol 24: O1-713, 1994 nt) rd29 salt, drought, cold Kasuga et al, Nature Biotechnol 18: 287-291, 1999 heat shock proteins, heat Barros et al, Plant Mol Biol 19 665–75, Including artificial 1992. Marrs et al, Dev Genet14: 27-41, promoters containing 1993. Schofflet al, Mol Gen Genet the heat shock 217: 246-53, 1989. element (HSE) SmHSP (small heat heat Waters et al, J Exp Bot 47: 325–338, 1996 shock proteins) wes120 cold Ouellete et al, FEBS Lett 423: 324–328, 1998 clf cold Kirch et al, Plant Mol Biol 33:897-909, 1997 Adh cold, drought, hypoxia Dolferus et al, Plant Physiol 105: 1075–87, 1994 pwsi18 salt and drought Joshee et al, Plant Cell Physiol 39: 64-72, 1998 cold Schneider et al, Plant Physiol 113: 335–45, 1997 US 2004/OO67506 A1 Apr. 8, 2004 19
TABLE 3-continued Exemplary plant-expressible promoters for use in the performance of the present invention Trg-31 drought Chaudhary et al, Plant Mol Biol 30: 1247–57, 1996 Osmotin osmotic Raghothama et al., Plant Mol Biol 23: 1117–28, 1993 LapA Wounding, WO99/03977 University of California/INRA enviromental
IV: EXEMPLARY PATHOGEN-INDUCBLE PROMOTERS
NAME PATHOGEN REFERENCE
RB7 Root-knot nematodes US576.0386 - North Carolina State (Meloidogyne spp.) University; Opperman et al, Science 263: 221-23, 1994 PR-1, 2, 3, 4, 5, 8, 11 fungal, viral, bacterial Ward et al, Plant Cell 3: 1085–1094, 1991; Reiss et al 1996; Label et al, Plant J 16: 223-233, 1998; Melchers et al, Plant J 5: 469-480, 1994: Lawton et al, Plant Mol Biol, 19: 735-743, 1992 HMG2 nematodes WO9503690 - Virginia Tech Intellectual Properties Inc. Abi3 Cyst nematodes unpublished (Heterodera spp.) ARM1 nematodes Barthels et al, Plant Cell 9: 2119–2134, 1997 WO 98/31822 - Plant Genetic Systems AttO728 nematodes Barthels et al, Plant Cell 9: 2119–2134, 1997 PCT/EP98/07761 Att1712 nematodes Barthels et al, Plant Cell 9, 2119-2134, 1997 PCT/EP98/07761 Gst1 Different types of Strittmatter et al, Mol Plant-Microbe Interact pathogens 9: 68-73, 1996 LEMMI nematodes WO92/21757 - Plant Genetic Systems CLE geminivirus PCT/EP99/03445 - CINESTAV PDF1.2 Fungal including Manners et al, Plant Mol Biol, 38: 1071-1080, Alternaria brassicicola 1998 and Botrytis cinerea Th2.1 Fungal - Fusarium Vignutelli et al, Plant J 14: 285-295, 1998 Oxysporum f.sp. matthiolae DBh226 CaOCle:S Bird and Wilson, Mol Plant-Microbe Interact 7:419–442, 1994 WO 95.322888 DBh28O CaOCle:S Bird and Wilson, Mol Plant-Microbe Interact 7:419–442, 1994 WO 95.322888 Cat? CaOCle:S Niebel et al, Mol Plant-Microbe Interact 8: 371-378, 1995 Tub CaOCle:S Aristizabal et al (1996), 8" International Congress on Plant-Microbe Interaction, Knoxville US B-29 SHSP CaOCle:S Fenoll et al (1997) In: Cellular and molecular aspects of plant-nematode interactions. Kluwer Academic, C. Fenoll, F. M. W. Grundler and S.A.. Ohl (Eds.), TSw12 CaOCle:S Fenoll et al (1997) In: Cellular and molecular aspects of plant-nematode Interactions. Kluwer Academic, C. Fenoll, F. M. W. Grundler and S.A.. Ohl (Eds.) Hs1(pro1) CaOCle:S WO 98/122335 - Jung NSLTP viral, fungal, bacterial Molina and Garcia-Olmedo FEBS Lett, 316: 119-122, 1993 RIP viral, fungal Tumer et al., Proc Natl Acad Sci USA 94:3866-3871, 1997 US 2004/OO67506 A1 Apr. 8, 2004 20
0190. Those skilled in the art will be aware of additional 0.195 Modulating, including lowering, the level of active promoter Sequences and terminator Sequences which may be gene products or of gene product activity can futhermore be Suitable for use in performing the invention. Such Sequences achieved by administering or exposing cells, tissues, organs may readily be used without any undue experimentation. or organisms to an inhibitor or activator of Said gene product 0191 In the context of the current invention, “ectopic or the activity thereof. Such inhibitors or activators include expression' or “ectopic overexpression' of a gene or a proteins (comprising e.g. proteinases and kinases) and protein are conferring to expression patterns and/or expres chemical compounds identified according to the current Sion levels of Said gene or protein normally not occurring invention as described Supra. under natural conditions. Ectopic expression can be 0196. In the context of the invention the term “agonist” achieved in a number of ways including operably linking of refers to a Substance that can be either a protagonist or an a coding Sequence encoding Said protein to an isolated antagonist, i.e. can have either positive or negative effects, homologous or heterologous promoter in order to create a can be an enhancer or an inhibitor or a modulator as well. chimeric gene and/or operably linking Said coding Sequence to its own isolated promoter (i.e. the unisolated promoter 0197). In the context of the current invention is envisaged naturally driving expression of said protein) in order to the downregulation of the expression of a LRR receptor-like create a recombinant gene duplication or gene multiplication kinase gene as defined higher. The invention further com effect. With “ectopic co-expression' is meant the ectopic prises downregulation of levels of a LRR receptor-like expression or ectopic overexpression of two or more genes kinase or of a LRR receptor-like kinase activity whereby or proteins. The same or, more preferably, different promot Said LRR receptor-like kinase has been defined Supra. erS are used to confer expression of Said genes or proteins. 0198 By “cell fate and/or plant development and/or plant 0.192 Preferably, the promoter sequence used in the con morphology and/or biochemistry and/or physiology” is text of the present invention is operably linked to a coding meant that one or more developmental and/or morphological Sequence or open reading frame (ORF) encoding a LRR and/or biochemical and/or physiological characteristics of a receptor-like kinase or a homolog, derivative and/or an plant is altered by the performance of one or more Steps immunologically active fragment thereof as defined Supra. pertaining to the invention described herein. 0193 “Dominant negative version or variant” refers to a 0199. “Cell fate” refers to the cell-type or cellular char mutant protein, which interferes with the activity of the acteristics of a particular cell that are produced during plant corresponding wild-type protein. development or a cellular process therefor, in particular 0194 “Downregulation of expression” as used herein during the cell cycle or as a consequence of a cell cycle means lowering levels of gene expression and/or levels of proceSS. active gene product and/or levels of gene product activity. 0200) “Plant development” or the term “plant develop Decreases in expression may be accomplished by e.g. the mental characteristic' or Similar term shall, when used addition of coding Sequences or parts thereof in a Sense herein, be taken to mean any cellular process of a plant that orientation (if resulting in co-Suppression) or in an antisense is involved in determining the developmental fate of a plant orientation relative to a promoter Sequence and furthermore cell, in particular the Specific tissue or organ type into which by e.g. insertion mutagenesis (e.g. T-DNA insertion or a progenitor cell will develop. Cellular processes relevant to transposon insertion) or by gene Silencing strategies as plant development will be known to those skilled in the art. described by e.g. Angell and Baulcombe (1998 Such processes include, for example, morphogenesis, pho WO9836083), Lowe et al. (1989–WO9853083), Lederer et tomorphogenesis, shoot development, root development, al. (1999–WO9915682) or Wang et al. (1999 vegetative development, reproductive development, Stem WO9953050). Genetic constructs aimed at silencing gene elongation, flowering, and regulatory mechanisms involved expression may have the nucleotide Sequence of Said gene in determining cell fate, in particular a process or regulatory (or one or more parts thereof) contained therein in a sense process involving the cell cycle. and/or antisense orientation relative to the promoter Sequence. Another method to downregulate gene expression 0201 “Plant morphology” or the term “plant morpho comprises the use of ribozymes, e.g. as described in Atkins logical characteristic' or Similar term will, when used et al. 1994 (WO9400012), Lenee et al. 1995 (WO9503404), herein, be understood by those skilled in the art to refer to Lutziger et al. 2000 (WO0000619), Prinsen et al. 1997 the external appearance of a plant, including any one or (WO9713865) and Scott et al. 1997 (WO9738116). Modu more Structural features or combination of Structural features lating, including lowering, the level of active gene products thereof. Such structural features include the shape, Size, or of gene product activity can be achieved by administering number, position, colour, texture, arrangement, and patter or exposing cells, tissues, organs or organisms to Said gene nation of any cell, tissue or organ or groups of cells, tissues product, a homolog, analogue, derivative and/or immuno or organs of a plant, including the root, Stem, leaf, shoot, logically active fragment thereof. Immunomodulation is petiole, trichome, flower, petal, Stigma, Style, Stamen, pol another example of a technique capable of downregulation len, ovule, Seed, embryo, endosperm, Seed coat, aleurone, levels of active gene product and/or of gene product activity fibre, fruit, cambium, wood, heartwood, parenchyma, aer and comprises administration of or exposing to or express enchyma, Sieve element, phloem or vascular tissue, amongst ing antibodies to Said gene product to or in cells, tissues, others. organs or organisms wherein levels of Said gene product 0202) “Plant biochemistry” or the term “plant biochemi and/or gene product activity are to be modulated. Such cal characteristic' or Similar term will, when used herein, be antibodies comprise “plantibodies', Single chain antibodies, understood by those skilled in the art to refer to the meta IgG antibodies and heavy chain camel antibodies as well as bolic and catalytic processes of a plant, including primary fragments thereof. and Secondary metabolism and the products thereof, includ US 2004/OO67506 A1 Apr. 8, 2004
ing any Small molecules, macromolecules or chemical com include 1 to 5 um gold spheres. The DNA construct may be pounds, Such as but not limited to Starches, Sugars, proteins, deposited on the microparticle by any Suitable technique, peptides, enzymes, hormones, growth factors, nucleic acid Such as by precipitation. molecules, celluloses, hemicelluloses, calloses, lectins, 0206. A whole plant may be regenerated from the trans fibres, pigments Such as anthocyanins, Vitamins, minerals, formed or transfected cell, in accordance with procedures micronutrients, or macronutrients, that are produced by well known in the art. Plant tissue capable of Subsequent plants. clonal propagation, whether by organogenesis or embryo genesis, may be transformed with a gene construct of the 0203 “Plant physiology” or the term “plant physiological present invention and a whole plant regenerated therefrom. characteristic' or Similar term will, when used herein, be The particular tissue chosen will vary depending on the understood to refer to the functional processes of a plant, clonal propagation Systems available for, and best Suited to, including developmental processes Such as growth, expan the particular species being transformed. Exemplary tissue Sion and differentiation, Sexual development, Sexual repro targets include leaf disks, pollen, embryos, cotyledons, duction, Seed Set, Seed development, grain filling, asexual hypocotyls, megagametophytes, callus tissue, existing mer reproduction, cell division, dormancy, germination, light istematic tissue (e.g., apical meristem, axillary buds, and adaptation, photosynthesis, leaf expansion, fiber production, root meristems), and induced meristem tissue (e.g., cotyle Secondary growth or wood production, amongst others, don meristem and hypocotyl meristem). responses of a plant to externally-applied factorS Such as metals, chemicals, hormones, growth factors, environment 0207. The term “organogenesis”, as used herein, means a and environmental stress factors (eg. anoxia, hypoxia, high process by which shoots and roots are developed Sequen temperature, low temperature, dehydration, light, daylength, tially from meristematic centers. flooding, salt, heavy metals, amongst others), including 0208. The term "embryogenesis”, as used herein, means adaptive responses of plants to Said externally-applied fac a process by which shoots and roots develop together in a torS. concerted fashion (not sequentially), whether from Somatic cells or gametes. 0204 Means for introducing recombinant DNA into plant 0209 Preferably, the plant is produced according to the tissue or cells include, but are not limited to, transformation inventive method is transfected or transformed with a using CaCl2 and variations thereof, in particular the method genetic Sequence, or amenable to the introduction of a described by Hanahan (1983), direct DNA uptake into protein, by any art-recognized means, Such as microprojec protoplasts (Krens et al., 1982; Paszkowski et al., 1984), tile bombardment, microinjection, Agrobactetrium-medi PEG-mediated uptake to protoplasts (Armstrong et al., 1990) ated transformation (including the “flower dip” transforma microparticle bombardment, electroporation (Fromm et al., tion method; Bechtold and Pelletier 1998, Trieu et al. 2000), 1985), microinjection of DNA (Crossway et al., 1986), protoplast fusion, or electroporation, amongst others. Most microparticle bombardment of tissue explants or cells preferably Said plant is produced by Agrobacterium-medi (Christou et al., 1988), vacuum-infiltration of tissue with ated transformation. nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described 0210. The “seedling” is the juvenile plant that arises from essentially by An et al.(1985), Dodds et al. (1985), Herrera the mature embryo after Seed germination. Estrella et al. (1983a, 1983b). Methods for transformation of monocotyledonous plants are well known in the art and 0211 With “differentiation of a cell” it is understood that include Agrobacterium-mediated transformation (Cheng et the cell developS unique features to be engaged for a specific al. 1997 WO9748814; Hansen 1998 WO9854961, Hiei function. Mostly differentiation is irreversible. et al. 1994 WO9400977; Hiei et al. 1998 WO9817813; 0212 Agrobacterium-mediated transformation or Rikiishi et al. 1999–WO9904618; Saito et al. 1995– agrolistic transformation of plants, yeast, moulds or fila WO9506722), microprojectile bombardment (Adams et al. mentous fungi is based on the transfer of part of the 1999. U.S. Pat. No. 5,969,213; Bowen et al. 1998 U.S. transformation vector Sequences, called the T-DNA, to the Pat. No. 5,736,369; Chang et al. 1994 - WO9413822; nucleus and on integration of Said T-DNA in the genome of Lundquist et al. 1999–U.S. Pat. No. 5,874.265/U.S. Pat. Said eukaryote. No. 5,990,390; Vasil and Vasil 1995 - U.S. Pat. No. 5,405, 765; Walker et al. 1999–U.S. Pat. No. 5,955,362), DNA 0213 With "Agrobacterium” is meant a member of the uptake (Eyalet al. 1993 - WO9318168), microinjection of Agrobacteriaceae, more preferably Agrobacterium or Rhizo Agrobacterium cells (von Holt 1994-DE4309203) and bacterium and most preferably Agrobacterium tumefaciens. sonication (Finer et al. 1997-U.S. Pat. No. 5,693,512). 0214) With “T-DNA", or transferred DNA, is meant that 0205 For microparticle bombardment of cells, a micro part of the transformation vector flanked by T-DNA borders particle is propelled into a cell to produce a transformed cell. which is, after activation of the Agrobacterium vir genes, Any Suitable ballistic cell transformation methodology and nicked at the T-DNA borders and is transferred as a single apparatus can be used in performing the present invention. stranded DNA to the nucleus of an eukaryotic cell. Exemplary apparatus and procedures are disclosed by Stomp 0215. When used herein, with “T-DNA borders”, et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. “T-DNA border region', or “border region” are meant either Pat. No. 4,945.050). When using ballistic transformation right T-DNA border (RB) or left T-DNA border (LB). Such procedures, the gene construct may incorporate a plasmid a border comprises a core Sequence flanked by a border inner capable of replicating in the cell to be transformed. region as part of the T-DNA flanking the border and/or a Examples of microparticles Suitable for use in Such Systems border outer region as part of the vector backbone flanking US 2004/OO67506 A1 Apr. 8, 2004 22 the border. The core Sequences comprise 22 bp in case of T-DNA. Said set of vir genes can be derived from either Octopine-type Vectors and 25 bp in case of nopaline-type Octopine-type or nopaline-type Tiplasmids or from both. vectors. The core Sequences in the right border region and left border region form imperfect repeats. Border core 0223 With “Super-binary transformation vector” is Sequences are indispensable for recognition and processing meant a binary transformation vector additionally carrying by the Agrobacterium nicking complex consisting of at least in the vector backbone region a Vir region of the Tiplasmid Vird 1 and Vird2. Core sequences flanking a T-DNA are pTiBo542 of the Super-virulent A. tumefaciens strain A281 sufficient to promote transfer of said T-DNA. However, (Hiei et al. 1994 EP0604662, Hieietal. 1995–EP0687730). efficiency of transformation using transformation vectors Super-binary transformation vectors are used in conjunction carrying Said T-DNA Solely flanked by Said core Sequences with a helper plasmid. is low. Border inner and outer regions are known to modu 0224. With “co-integrate transformation vector” is meant late efficiency of T-DNA transfer (Wang et al. 1987). One a T-DNA vector at least comprising: a T-DNA region com element enhancing T-DNA transfer has been characterized prising at least one gene of interest and/or at least one and resides in the right border outer region and is called Selectable marker active in plants, and a vector backbone overdrive (Peralta et al. 1986, van Haaren et al. 1987). region comprising at least origins of replication active in Escherichia coli and Agrobacterium, and markers for Selec 0216) With “T-DNA transformation vector” or “T-DNA tion in E. coli and Agrobacterium, and a Set of Vir genes vector” is meant any vector encompassing a T-DNA necessary for enabling transfer of the T-DNA. sequence flanked by a right and left T-DNA border consist ing of at least the right and left border core Sequences, 0225. The T-DNA borders and said set of vir genes of a respectively, and used for transformation of any eukaryotic said T-DNA vector can be derived from either octopine-type cell. or nopaline-type Tiplasmids or from both. 0217. With “T-DNA vector backbone sequence” or 0226. With “Ri-derived plant transformation vector” is “T-DNA vector backbone sequences” is meant all DNA of a meant a binary transformation vector in which the T-DNA T-DNA containing vector that lies outside of the T-DNA borders are derived from a Ti plasmid and said binary borders and, more Specifically, outside the nicking Sites of transformation vector being used in conjunction with a the border core imperfect repeats. helper Ri-plasmid carrying the necessary Set of Vir genes. 0218. The current invention includes optimized T-DNA 0227 AS used herein, the term “selectable marker gene” vectorS Such that vector backbone integration in the genome or “selectable marker” or “marker for selection' includes of a eukaryotic cell is minimized or absent. With “optimized any gene which confers a phenotype on a cell in which it is T-DNA vector” is meant a T-DNA vector designed either to expressed to facilitate the identification and/or Selection of decrease or abolish transfer of Vector backbone Sequences to cells which are transfected or transformed with a gene the genome of a eukaryotic cell. Such T-DNA vectors are construct of the invention or a derivative thereof. Suitable known to the one familiar with the art and include those Selectable marker genes contemplated herein include the described by Hanson et al. (1999) and by Stuiver et al. ampicillin resistance (Amp', tetracycline resistance gene (Tc), bacterial kanamycin resistance gene (Kan'), phosphi (1999 WO9901563). nothricin resistance gene, neomycin phosphotransferase 0219. The current invention clearly considers the inclu gene (npfll), hygromycin resistance gene, f-glucuronidase Sion of a DNA sequence comprising the promoter Sequence (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, of the present invention encoding a LRR receptor-like green fluorescent protein (gfp) gene (Haseloff et al., 1997), kinase, homolog, derivative or immunologically active frag and luciferase gene, amongst others. ment thereof as defined supra, in any T-DNA vector com prising binary transformation vectors, Super-binary transfor 0228. With “agrolistics”, “agrolistic transformation” or mation vectors, co-integrate transformation vectors, “agrolistic transfer” is meant here a transformation method Ri-derived transformation vectors as well as in T-DNA combining features of Agrobacterium-mediated transforma carrying vectors used in agrolistic transformation. tion and of biolistic DNA delivery. As such, a T-DNA containing target plasmid is co-delivered with DNA/RNA 0220. With “binary transformation vector” is meant a enabling in plant a production of Vird 1 and Vird2 with or T-DNA transformation vector comprising: a T-DNA region without VirE2 (Hansen and Chilton 1996; Hansen et al. comprising at least one gene of interest and/or at least one 1997; Hansen and Chilton 1997–WO9712046). The Selectable marker active in the eukaryotic cell to be trans present invention further describes an approach to remove formed; and a vector backbone region comprising at least from transformed cells a stably integrated foreign DNA origins of replication active in E. coli and Agrobacterium Sequence by recombination involving a recombinase and and markers for Selection in E. coli and Agrobacterium. recombination sites. 0221) The T-DNA borders of a binary transformation 0229. With “foreign DNA” is meant any DNA sequence vector can be derived from Octopine-type or nopaline-type that is introduced in the host's genome by recombinant Tiplasmids or from both. The T-DNA of a binary vector is techniques. Said foreign DNA includes e.g. a T-DNA only transferred to a eukaryotic cell in conjunction with a Sequence or a part thereof Such as the T-DNA sequence helper plasmid. Also known in the art are multiple binary comprising the Selectable marker in an expressible format. vector Agrobacterium Strains for efficient co-transformation Foreign DNA furthermore include intervening DNA of plants (Bidney and Scelonge 2000–WO0018939). Sequences as defined Supra. 0222. With “helper plasmid' is meant a plasmid that is 0230. With “recombination event' is meant either a site Stably maintained in Agrobacterium and is at least carrying Specific recombination event or a recombination event the Set of Vir genes necessary for enabling transfer of the effected by transposon jumping. US 2004/OO67506 A1 Apr. 8, 2004
0231. With “recombinase” is meant either a site-specific ing or through croSS-pollination. Alternatively, a Substan recombinase or a transposase. tially purified recombinase protein could be introduced directly into the eukaryotic cell, e.g. by micro-injection or 0232. With “recombination' site is meant either site particle bombardment. Typically, the Site-specific recombi Specific recombination sites or transposon border Sequences. nase coding region will be operably linked to regulatory 0233 With “site specific recombination event' is meant Sequences enabling expression of the Site-specific recombi an event catalyzed by a System generally consisting of three nase in the eukaryotic cell. elements: a pair of DNA sequences (the site-specific recom bination sequences or sites) and a specific enzyme (the 0235 With “recombination event effected by transposon Site-specific recombinase). The site-specific recombinase jumping” or “transposase-mediated recombination' is meant catalyzes a recombination reaction only between two site a recombination event catalyzed by a System consisting of Specific recombination Sequences depending on the orien three elements: a pair of DNA sequences (the transposon tation of the site-specific recombination Sequences. border Sequences) and a specific enzyme (the transposase). Sequences intervening between two site-specific recombi The transposase catalyzes a recombination reaction only nation sites will be inverted in the presence of the Site between two transposon border Sequences which are Specific recombinase when the Site-specific recombination arranged as inverted repeats. A number of different trans Sequences are oriented in opposite directions relative to one poSon/transposase Systems can be used including but not another (i.e. inverted repeats). If the site-specific recombi limited to the DS/Ac system, the Spm system and the Mu nation Sequences are oriented in the same direction relative System. These Systems originate from corn but it has been to one another (i.e. direct repeats), then any intervening shown that at least the DS/Ac and the Spm system also Sequences will be deleted upon interaction with the Site function in other plants (Fedoroffet al. 1993, Schlappi et al. Specific recombinase. Thus, if the Site-specific recombina 1993, Van Sluys et al. 1987). Preferred are the Ds- and the tion Sequences are present as direct repeats at both ends of Spm-type transposons which are delineated by 11 bp- and 13 a foreign DNA sequence integrated into a eukaryotic bp-border Sequences, respectively. genome, Such integration of Said Sequences can Subse 0236 Although the transposon border sequences must be quently be reversed by interaction of the Site-specific recom linked to the ends of the DNA to be excised, the gene bination Sequences with the corresponding site Specific encoding the transposase may be located elsewhere. For recombinase. A number of different site specific recombi example, the recombinase gene could already be present in nase Systems can be used including but not limited to the the eukaryotes DNA or could be supplied by a later intro Cre/lox system of bacteriophage P1, the FLP/FRT system of duced DNA fragment either introduced directly into cells, yeast, the Gin recombinase of phage Mu, the Pin recombi through crossing or through cross-pollination. Alternatively, nase of E. coli, the PinB, PinD and PinF from Shigella, and a Substantially purified transposase protein could be intro the R/RS system of Zygosaccharomyces rouxil. Recombi duced directly into cells, e.g. by microinjection or by particle nases generally are integrases, resolvases or flippases. Also bombardment. dual-specific recombinases can be used in conjunction with direct or indirect repeats of two different Site-specific recom 0237 As part of the current invention, transposon border bination sites corresponding to the dual-specific recombi Sequences are included in a foreign DNA sequence Such that nase (Baszczynski et al. 1999 - WO9925840). The pre they lie outside said DNA sequence and transform said DNA ferred site-specific recombinase Systems are the into a transposon-like entity that can move by the action of bacteriophage P1 Cre/lox, the yeast FLP/FRT and the Z. a transposase. rouxi R/RS systems. In these Systems a recombinase (Cre, 0238 AS transposons often reintegrate at another locus of FLP or R) interact specifically with its respective site the host's genome, Segregation of the progeny of the hosts Specific recombination sequence (lox, FRT, or RS respec in which the transposase was allowed to act might be tively) to invert or excise the intervening sequences. The necessary to Separate transformed hosts containing e.g. only Site-specific recombination Sequences for each of these two the transposon footprint and transformed hosts Still contain systems are relatively short (34 bp for 10x and 47 bp for ing the foreign DNA. FRT). Some of these systems have already been used with high efficiency in plants such as tobacco (Dale et al. 1990, 0239). In performing the present invention, the genetic Onouchi et al. 1991, Sugita et al. 2000) and Arabidopsis element is preferably induced to mobilize, Such as, for (Osbome et al. 1995, Onouchi et al. 1995). Site-specific example, by the expression of a recombinase protein in the recombination Systems have many applications in plant cell which contacts the integration Site of the genetic ele ment and facilitates a recombination event therein, excising molecular biology including methods for control of homolo the genetic element completely, or alternatively, leaving a gous recombination (e.g. Hodges et al. 1996-U.S. Pat. No. “footprint', generally of about 20 nucleotides in length or 5,527,695), for targeted insertion, gene Stacking, etc. greater, at the original integration site. Those hosts and host (Baszczynski et al. 1999–WO9925821) and for resolution parts that have been produced according to the inventive of complex T-DNA integration patterns or for excision of a method can be identified by standard nucleic acid hybrid selectable marker (Owet al. 1999–WO9923202). ization and/or amplification techniques to detect the pres 0234. Although the site-specific recombination ence of the mobilizable genetic element or a gene construct sequences must be linked to the ends of the DNA to be comprising the same. Alternatively, in the case of trans excised or to be inverted, the gene encoding the Site specific formed host cells, tissues, and hosts wherein the mobilizable recombinase may be located elsewhere. For example, the genetic element has been excised, it is possible to detect a recombinase gene could already be present in the eukary footprint in the genome of the host which has been left ote's DNA or could be supplied by a later introduced DNA following the excision event, using Such techniques. AS used fragment either introduced directly into cells, through croSS herein, the term “footprint” shall be taken to refer to any US 2004/OO67506 A1 Apr. 8, 2004 24 derivative of a mobilizable genetic element or gene con dependent kinases (CDK), cyclin dependent kinase inhibi Struct comprising the same as described herein which is tors (CKI), D, E and A cyclins, E2F and DP transcription produced by excision, deletion or other removal of the factors, pocket proteins, CDC7/DBF4 kinase, CDC6, mobilizable genetic element from the genome of a cell MCM2-7, Orc proteins, cdc45, components of SCF ubiq transformed previously with Said gene construct. A footprint uitin ligase, PCNA, DNA-polymerase. generally comprises at least a single copy of the recombi nation loci or transposon used to promote excision. How 0245. The term “cell cycle control protein' include ever, a footprint may comprise additional Sequences derived cyclins A, B, C, D and E including CYCA1;1, CYCA2;1, from the gene construct, for example nucleotide Sequences CYCA3;1, CYCB1;1, CYCB1;2, CYC B2:2, CYCD1;1, derived from the left border sequence, right border CYCD2:1, CYCD3;1, and CYCD4;1 (Evans et al. 1983; Sequence, origin of replication, recombinase-encoding or Francis et al. 1998; Labbe et al. 1989; Murray & Kirschner transposase-encoding Sequence if used, or other vector 1989; Renaudin et al. 1996; Soni et al. 1995; Sorrell et al. derived nucleotide Sequences. Accordingly, a footprint is 1999; Swenson, Farrell, & Ruderman 1986) cyclin depen identifiable according to the nucleotide Sequence of the dent kinase inhibitor (CKI) proteins such as ICK1 (Wang, recombination locus or transposon of the gene construct Fowke, & Crosby 1997), FL39, FL66, FL67 (PCT/EP98/ used, Such as, for example, a Sequence of nucleotides 05895), Sic1, Far1, Rum1, p21, p27, p57, p16, p15, pl8, p 19 corresponding or complementary to a lox site, frt Site or RS (Elledge 1996; Pines 1995), p.14 and p14ARF; p13suc1 or Site. CKS1At (De Veyider et al. 1997; Hayles & Nurse 1986) and nim-1 (Fantes 1989; Russell & Nurse 1986; Russell & Nurse 0240 The term “cell cycle” means the cyclic biochemical 1987b; Russell & Nurse 1987a; Russell & Nurse 1986) and structural events associated with growth and with divi homologues of Cdc2 such as Cdc2MsB (Hirt et al. 1993) Sion of cells, and in particular with the regulation of the CdcMs kinase (Bogre et al. 1997) cdc2 T14Y15 phos replication of DNA and mitosis. Cell cycle includes phases phatases Such as Cdc25 protein phosphatase or p80cdc25 called: G0, Gap 1 (G1), DNA synthesis (S), Gap2 (G2), and (Bell et al. 1993; Elledge 1996; Kumagai & Dunphy 1991; mitosis (M). Normally these four phases occur Sequentially, Russell et al. 1986) and Pyp3 (Elledge 1996) cdc2 protein however, the cell cycle also includes modified cycles kinase or p34.cdc2 (Colasanti, Tyers, & Sundaresan 1991; wherein one or more phases are absent resulting in modified Feiler & Jacobs 1990; Hirt et al. 1991; John, Sek, & Lee cell cycle Such as endomitosis, acytokinesis, polyploidy, 1989; Lee & Nurse 1987; Nurse & Bissett 1981; Ormrod et polyteny, and endoreduplication. al. 1993) cdc2a protein kinase (Hemerly et al. 1993) cdc2 0241 The term “cell cycle interacting protein”, “cell T14Y15 kinases such as wee1 or p107wee1 (Elledge 1996; cycle protein’ or “cell cycle control protein’ as denoted Russell et al. 1986; Russell et al. 1987b; Russell et al. 1987a; herein means a protein which exerts control on or regulates Sun et al. 1999) mik1 (Lundgren et al. 1991) and myt1 or is required for the cell cycle or part thereof of a cell, (Elledge 1996); cdc2 T161 kinases such as Cak and Civ tissue, organ or whole organism and/or DNA replication. It (Elledge 1996); cdc2 T161 phosphatases such as Kap1 may also be capable of binding to, regulating or being (Elledge 1996); cdc28 protein kinase or p34.cdc28 (Nasmyth regulated by cyclindependent kinases or their Subunits. The 1993; Reed, Hadwiger, & Lorincz 1985) p40MO15 (Fesquet term also includes peptides, polypeptides, fragments, Vari et al. 1993; Poon et al. 1993) chk1 kinase (Zeng et al. 1998) ant, homologs, alleles or precursors (eg preproproteins or cds1 kinase (Zeng et al. 1998) growth-associated H1 kinase preproteins) thereof. (Labbe et al. 1989; Lake & Salzman 1972; Langan 1978; Zeng et al. 1998) MAP kinases described by (Binarova et al. 0242 Cell cycle control proteins and their role in regu 1998; Bogreet al. 1999; Calderini et al. 1998; Wilson et al. lating the cell cycle of eukaryotic organisms are reviewed in 1999). detail by John (1981) and the contributing papers therein (Norbury & Nurse 1992; Nurse 1990; Ormrod & Francis 0246. Other cell cycle control proteins that are involved 1993) and the contributing papers therein (Doerner et al. in cyclin D-mediated entry of cells into G1 from G0 include 1996) which are incorporated by reference.(Elledge 1996; pRb (Huntley et al. 1998; Xie et al. 1996) E2F, RIP, MCM7 Francis, Francis & Halford 1995; Hirt et al. 1991; Mironov and potentially the pRb-like proteins p107 and p130. et al. 1999) 0247. Other cell cycle control proteins that are involved 0243 The term “cell cycle control genes” refers to any in the formation of a pre-replicative complex at one or more gene or mutant thereof which exerts control on or are origins of replication, Such as, but not limited to, ORC, required for chromosomal DNA synthesis and for mitosis CDC6, CDC14, RPA and MCM proteins or in the regulation (preprophase band, nuclear envelope, spindle formation, of formation of this pre-replicative complex, Such as, but not chromosome condensation, chromosome Segregation, for limited to, the CDC7, DBF4 and MBF proteins. mation of new nuclei, formation of phragmoplast, duplica tion of microtubule-organizing center, etc) meiosis, cytoki 0248 For the present purpose, the term “cell cycle con nesis, cell growth, endoreduplication, cell cycle control trol protein” shall further be taken to include any one or more of those proteins that are involved in the turnover of genes are also all genes exerting control on the above: any other cell cycle control protein, or in regulating the homologs of CDKs, cyclins, E2Fs, Rb, CKI, Cks, and also half-life of said other cell cycle control protein. The term any genes which interfere with the above, cyclin D, cdc25, “protein turnover” is to include all biochemical modifica Wee 1, Nim1, MAP kinases, etc. tions of a protein leading to the physical or functional 0244 More specifically, cell cycle control genes are all removal of Said protein. Although not limited to these, genes involved in the control of entry and progression examples of Such modifications are phosphorylation, ubiq through S phase. They include, not exclusively, genes uitination and proteolysis. Particularly preferred proteins expressing “cell cycle control proteins' Such as cyclin which are involved in the proteolysis of one or more of any US 2004/OO67506 A1 Apr. 8, 2004
other of the above-mentioned cell cycle control proteins or the original Sequence referred to, while the maximum size include the yeast-derived and animal-derived proteins, is not critical. In Some applications, the maximum size Skp1, Skp2, Rub1, Cdc20, cullins, CDC23, CDC27, usually is not Substantially greater than that required to CDC16, and plant-derived homologues thereof (Cohen-Fix provide the desired activity and/or function(s) of the original & Koshland 1997; Hochstrasser 1998; Krek 1998; Lisztwan Sequence. Typically, the truncated amino acid or nucleotide et al. 1998) and Plesse et all in (Francis et al. 1998)). Sequence will range from about 5 to about 60 amino acids in 0249 For the present, purpose, the term “cell cycle length. More typically, however, the Sequence will be a control genes' shall further be taken to include any one or maximum of about 50 amino acids in lenght, preferably a more of those gene that are involved in the transcriptional maximum of about 60 amino acids. It is usually desirable to regulation of cell cycle control gene expression Such as Select Sequences of at least about 10, 12 or 15 amino acids transcription factors and upstream Signal proteins. Addi or nucleotides, up to a maximum of about 20 or 25 amino tional cell cycle control genes are not excluded. acids or nucleotides. 0250 For the present purpose, the term “cell cycle con 0256 Furthermore, folding simulations and computer trol genes' shall further be taken to include any cell cycle redesign of Structural motifs of the protein of the invention control gene or mutant thereof, which is affected by envi can be performed using appropriate computer programs ronmental Signals Such as for instance StreSS, nutrients, (Olszewski et al. 1996, Hoffman et al. 1995). Computer pathogens, or by intrinsic Signals Such as the animal mito modeling of protein folding can be used for the conforma gens or the plant hormones (auxins, cytokinins, ethylene, tional and energetic analysis of detailed peptide and protein gibberellic acid, abscisic acid and brassinosteroids). models (Monge et al. 1995, Renouf et al. 1995). In particu 0251 The term “cell cycle progression” refers to the lar, the appropriate programs can be used for the identifi process of passing through the different cell cycle phases. cation of interactive sites of the LRR receptor-like kinase of The term "cell cycle progression rate” accordingly refers to the present invention by computer assistant Searches for the Speed at which Said cell cycle phases are run through or complementary peptide Sequences (Fassina and Melli 1994). the time spans required to complete Said cell cycle phases. Further appropriate computer Systems for the design of protein and peptides are described in the prior art, for 0252 With “pathogen' is meant those organisms that example in Berry and Brenner (1994), Wodak (1987), Pabo have a negative effect on the physiological State of the plant and Suchanek (1986). The results obtained form the above or a part thereof. Some pathogens are for instanc viruses, described computer analysis can be used for, e.g. the prepa bacteria, fungi, and parasitic plants, with plant "pests” is ration of peptidomimetics of the protein of the invention or meant the group of nematodes as well as insects, which are fragments thereof. Such pseudopeptide analogues of the able to attack and to damage the plant. "Plant cell’ com natural amino acid Sequence of the protein may very effi prises any cell derived from any plant and existing in culture ciently mimic the parent protein (Benkirane et al. 1996). For as a Single cell, a group of cells or a callus. A plant cell may example, incorporation of easily available achiral (D-amino also be any cell in a developing or mature plant in culture or acid residues into a protein of the invention or a fragment growing in nature. thereof results in the substitution of amino bonds by poly 0253 “Plants' comprises all plants, including monocoty methylene units of an aliphatic chain, thereby providing a ledonous and dicotyledonous plants. convenient strategy for constructing a peptidomimetic (Ban erjee et al. 1996). Superactive peptidomimetic analogues of 0254. With “yeast two-hybrid assay” is meant an assay Small peptide hormones in other Systems are described in the that is based on the observation that many eukaryotic prior art (Zhang et al. 1996). Appropriate peptidomimetics transcription factors comprise two domains, a DNA-binding of the protein of the present invention can also be identified domain (DB) and an activation domain (AD) which, when by the Synthesis of peptidomimetic combinatorial libraries physically separated (i.e. disruption of the covalent linkage) through Successive amine alkylation and testing the resulting do not effectuate target gene expression. Two proteins able compounds, e.g., for their binding, kinase inhibitory and/or to interact physically with one of said proteins fused to DB immunlogical properties. Methods for the generation and and the other of said proteins fused to AD will re-unite the use of peptidomimetic combinatioral libraries are described DB and AD domains of the transcription factor resulting in in the prior art, for example in Ostresh et al. (1996) and target gen expression. The target gene in the yeast two Dorner et al. (1996). hybrid assay is usually a reporter gene Such as the B-galac tosidase gene. Interaction between protein partners in the 0257) Furthermore, a three-dimensional and/or crystallo yeast two-hybrid assay can thus be quantified by measuring graphic structure of the protein of the invention can be used the activity of the reporter gene product (Bartel and Fields for the design of peptidomimetic inhibitors of the biological 1997). Alternatively, a mammalian two-hybrid system can activity of the protein of the invention (Rose et al. 1996, be used which includes e.g. a chimeric green fluorescent Ruterber et al. 1996). protein encoding reporter gene (Shioda et al. 2000). Yet 0258 To align sequences we used the Clustal V method another alternative consists of a bacterial two-hybrid System described in Higgins and Sharp (Higgins D G & Sharp PM, using e.g. HIS as reporter gene (Joung et al. 2000). Comput Appl BioSci. 1989 April; 5(2) 151-3). The Clustal 0255 The term “fragment of a sequence” or “part of a Method groupS. Sequences into clusters by examining Sequence” means a truncated Sequence of the original Sequence distances between all pairs. Clusters are aligned as Sequence referred to. The truncated Sequence (nucleic acid pairs then collectively as Sequence groups to produce the or protein sequence) can vary widely in length; the mini overall alignment. After the multiple alignment is com mum size being a Sequence of Sufficient size to provide a pleted, a neighbor-joining method is employed to recon Sequence with at least a comparable function and/or activity Struct phylogeny for the putative alignment. US 2004/OO67506 A1 Apr. 8, 2004 26
0259 1. Residue Weight Table represents the distance between Sequence pairs, while the 0260 The Clustal method uses weight tables to construct units at the bottom of the tree indicate the number of multiple alignments. Residue weight tables are used in Substitution events. Scoring protein and nucleotide alignments So that slightly 0276 The phylogenetic tree gives a first estimate of the mismatched residues, Such as lie VS. Val amino acids or GVS. relationships between homologous Sequences. It is highly R nucleotides, are not Scored the Same as total mismatches. recommended that your data be evaluated using additional methods before final conclusions are made about evolution 0261) If your sequences are protein, or a mixture of ary relationships. protein and DNA, choose between PAM250, PAM 100, Structural and Identity. 0277 You can switch the view between Balanced 0262) 1... iPAM250 is recommended for the Clustal Branches (phenogram) or Unbalanced Branches (cla method and is ideal for longer Sequences or highly diverged dogram). A phenogram averages the distances between ancestors in the tree. Dotted lines indicate a negative branch Sequences. length, which may be a byproduct of averaging. In the 0263. 2. PAM 100 is also used with the Clustal method cladogram, branch distances correspond to Sequence diver and is most accurate for shorter Sequences or Sequences gence. having little divergence. 0278. The compounds to be obtained or identified in the 0264 3. iStructural is recommended for the Jotun Hein methods of the invention can be compounds that are able to method and gives higher Scores to residues that are chemi bind to any of the nucleic acids, peptides or proteins of the cally and Spatially Similar. invention. Other interesting compounds to be identified are compounds that modulate the expression of the genes or the 0265. 4. Identity may be used with Clustal or Jotun Hein proteins of the invention in Such a way that either the methods, but is recommended only for the Clustal method. expression of Said gene or protein is enhanced or decreased The table Scores non-identities as Zero, and identities as a by the action of Said compound. Alternatively the compound default value of 10. can exert his action by directly or indirectly enhancing or 0266 2. Method Parameters decreasing the activity of any of the proteins of the inven tion. 0267 A. Pairwise Alignment Parameters may include K-tuple, Gap Penalty, Window and Diagonals Saved: 0279 Said compound or plurality of compounds may be comprised in, for example, Samples, e.g., cell extracts from, 0268 1. Specify the K-tuple, expressed as the units e.g., plants, animals or microorganisms. Furthermore, Said length in nucleotides. Any Stretch of K or more compound(s) may be known in the art but hitherto not adjacent nucleotides that form an exact match in both known to be capable of Suppressing or activating cell cycle Sequences is a K-tuple. A lower K-tuple will find interacting proteins. The reaction mixture may be a cell free Smaller matching regions, a higher value will find extract of may comprise a cell or tissue culture. Suitable Set fewer, but better, matching regions of Similarity. ups for the method of the invention are known to the perSon skilled in the art and are, for example, generally described in 0269 2. Type in the Gap Penalty, the amount deducted Alberts et al., Molecular Biology of the Cell, third edition from the Score for each gap in the alignment. Gaps of (1994), in particular Chapter 17. The plurality of compounds different sizes carry the Same penalty. may be, e.g., added to the reaction mixture, culture medium 0270. 3. Specify the Window, the range the program or injected into the cell. Searches for all possible or alternate K-tuples. An extremely 0280) If a sample containing a compound or a plurality of high value may slow an alignment by considering Subalign compounds is identified in the method of the invention, then ments far from the main diagonal. it is either possible to isolate the compound form the original 0271 4. Type in the number of Diagonals Saved. In the Sample identified as containing the compound capable of pairwise calculation, the best Scoring diagonals for each pair acting as an agonist, or one can further Subdivide the original in the matrix are Saved and used in the clustering and Scoring Sample, for example, if it consists of a plurality of different of ancestors. compounds, So as to reduce the number of different Sub stances per Sample and repeat the method with the Subdivi 0272 B. Multiple Alignment Parameters include Gap Sions of the original Sample. Depending on the complexity Penalty and Gap Length Penalty. of the Samples, the Steps described above can be performed Several times, preferably until the Sample identified accord 0273 1. Specify the Gap Penalty, the amount deducted ing to the method of the invention only comprises a limited from the Score for each gap in the alignment. Gaps of number of or only one substance(s). Preferably said sample different sizes carry the Same penalty. comprises Substances or Similar chemical and/or physical 0274) 2. Specify the Gap Length Penalty, the value which properties, and most preferably Said Substances are identical. will be deducted from the alignment score after first multi Preferably, the compound identified according to the above plying it by the number of gaps. Longer gaps have a greater described method or its derivative is further formulated in a penalty than shorter gaps. form Suitable for the application in plant breeding or plant cell and tissue culture. 0275 Whenever in the current description reference is made to a "phylogenetic tree', this phylogenetic tree is used 0281. The invention, now being generally described, will to view phylogenetic relationships compatible with multiple be more readily understood by reference to the following Sequence alignments. The length of each pair of branches examples, which are included merely for purposes of illus US 2004/OO67506 A1 Apr. 8, 2004 27 tration of certain aspects and embodiments of the present 0295) This gene's open reading frame starts at the posi invention and are not intended to limit the invention. tion 1759-1757 with the ATG (bold), contains two introns, and ends at position 350-348 with a TGA codon (bold). A DESCRIPTION OF FIGURES homology Search of the protein Sequence of this antisense gene in the RCH1 prom against the protein database with the 0282 FIG. 1. Models for stem-cell maintenance in the program BLASTP 2.0.8 (Altschul et al. 1997) produced a root and shoot meristem. hypothetical protein (Accession number AC002392, clone 0283 (a) In the root meristem, the quiescent center (QC) T20K24.8) that is a putative anthranilate N-hydroxycin functions as an organizing center. Those cells in immediate namoyl/benzoyltransferase from Arabidopsis thaliana. Both contact with the QC are stem cells (dark shading), which are sequences show 38% identity and 54% of their amino acids initials for all cell files: endodermis (end), cortex (cort), are homologous. epidermis (epi), lateral root cap (Irc) and columella (col). (b) 0296 FIG.7. Promoteractivity of the RCH1 promoter in the shoot meristem, WUS is functionally equivalent to the GUS fusion construct in the Arabidopsis thaliana roots. QC in the root meristem in maintaining the presence of Stem cells (dark Shading). Long-range Signaling for Stem cell 0297 Seedlings of different age were grown and stained maintenance is also postulated for ZLL, which is expressed with Glucuronidase (GUS). The expression of the GUS in the vascular cells. CLV3 is expressed in the presumptive reporter gene can be seen in the root meristem of the main stem cells that the initials for the L1 (epidermis), L2 and the lateral roots, as well as in the vascular tissue of the (Subepidermal tissues, i.e. mesophyll cells) and L3 (Subepi root and the lateral root primordia. (A) 1 day old Seeding dermal tissues, i.e. vascular cells) tissue layers (Schnittger, Stained over night, (B) higher magnification of A, (C) 2 days Schellmann, & Hulskamp 1999). CLV1 is found in the L3 old seedling stained for 5 hours, (D) main root from 1 day tunica layer of the central Zone. old Seedling stained for 1 hour, (E) lateral root primordia from 1 week old seedling stained for 5 hours, (F, G, H) 0284 FIG. 2. List of the degenerative primers used for lateral roots from an 8 days old Seedling Stained for 5 hours. RT-PCR of root-specific members of the LRR receptor kinase family. 0298 FIG. 8. The nucleotide sequence of the RCH1 0285 FIG. 3. Differential RT-PCR on hypocotyl/cotyle gene. don (C) and roottip (R)RNA. 0299. This fragment of the genomic RCH1 clone is about 3960 base pairs long and is identified as SEQID NO3. Two 0286 The PCR fragments obtained upon amplification of putative ATG start codons of the RCH1 protein on position R and C cl)NAS with degenerate primers are separated on 1-3 and on position 13-15 could be identified and are agarose gel. DNA fragments of different size can be detected represented in bold. Also the TMAA stopcodon on position in the R compared to C. PCRS. 3958-3960 is represented in bold. There is a putative intron 0287 M represents the 100 bp marker (Boehringer). C estimated to range from position 2900 (CAGGTA) to and R indicated the origin of the cDNA sample used in the position 3452 (CAGAAT) with the program http://ww PCR and the numbers below indicate the primer combina w.CBS.DTU.DK/services/NetGene2. The putative splice tion used, e.g. C. represents hypocotyl/cotyledon cDNA Sites are underlined. amplified with primers LRR2 and KIN3. 0300 FIG. 9. The amino acid sequence of the open 0288 FIG. 4. RT-PCR analysis of RCH1 expression. reading frame encoding RCH1 protein. 0289 Total RNA was isolated from flower (F), hypo 0301 Two putative start methionins of the RCH1 protein cotyl/cotyledon (C) and roottips (R) and RCH1 mRNA was on position 1 and on position 4 could be identified. An ORF amplified for 26 cycles with RT-PCR using the RCH1F and of 1135 amino acids is identified and represented in SEQ ID RCH1R primers. As a control for cDNA quality and quantity NO 4. The ORF of 1131 amino acids and starting at the as well as relative expression level, ubiquitin (Ubi) mRNA methionine on position 4 was amplified for 20 cycles under the same conditions using the UBIF and UBIR primers. 0290 FIG. 5. The nucleotide sequence of the RCH1 operOn. (M) 0291. This figure shows het nucleotide sequence of SEQ ID NO 1 that contains the genomic RCH1 clone. 0302) corresponds with SEQ ID NO 19. 0292 FIG. 6. The nucleotide sequence of the RCH1 0303) The boxed peptide promoter region. 0293. This fragment of the genomic RCH1 clone is about 3498 base pairs and is identified as SEQ ID NO 2. Two RYKIILGAAOGLAYLHHDCVPPIVH putative ATG start codons of the RCH1 protein on position 3479-3481 and on position 3491-3493 could be identified and are represented in bold. The underlined Sequence cor 0304 corresponds with SEQ ID NO 5. responds to SEQ ID NO 18. 0305 FIG. 10. Amino acid alignment of the RCH1 0294. It is also known that there is a gene in the reverse protein the Arabidopsis thaliana CLV1 receptor kinase like orientation in this promoter region. protein. US 2004/OO67506 A1 Apr. 8, 2004 28
0306 The amino acid sequences of the RCH1 and of 0318 FIG. 17. Amino acid sequence of RCH1 homo CLV1 (Genbank accession number AAB58929) were logues of Table 4 aligned using the program GAP as described in example 3. Match display thresholds for the alignment(s) are: =IDEN EXAMPLES TITY:=2,..-1. 0319 Unless stated otherwise in the Examples, all recombinant DNA techniques are performed according to 0307 FIG. 11. Phylogenetic tree of CLV1 homologues. protocols as described in Sambrook et al. (1989), Molecular 0308 The novel RCH1 sequence was aligned with the Cloning: A Laboratory Manual. Cold Spring Harbor Labo CLV1 (Genbank accession number U96879.1), the known ratory Press, NY or in Volumes 1 and 2 of Ausubel et al., CLV1 homologues BRI1 (Genbank accession number (1994), Current Protocols in Molecular Biology, Current AF017056), HAE (GenPept accession number S27756) and Protocols. Standard materials and methods for plant molecu ER (Genbank accession number D83257) as well as with 4 lar work are described in Plant Molecular Biology Labfase other related proteins that were retrieved from the public (1993) by R. D. D. Croy, jointly published by BIOS Scien database searched with the RCH1 sequence using the BlastP tific Publications Ltd. (UK) and Blackwell Scientific Pub program. The multiple alignment and phylogenetic tree of lications (UK). Said Sequences were calculated with the Clustal V method as Example 1 described in example 4. Based on the Sequence, RCH1 is more homologue to CLV1 than other known CLV-homo Isolation of the RCH1 Operon From Root Tissue of logues. Arabidopsis thaliana 0309 FIG. 12. Phylogenetic tree of LRR receptor like 0320 Plant organ development depends on the presence kinases of distally positioned groups of continuously dividing cells, the shoot and root apical meristems. Laser ablation Studies 0310. The novel RCH1 sequence was aligned with CLV1, Suggest that a balance between Signals for proper differen its homologues BRI, HAE, ER and other LRR receptor like tiation and Short range Signals from the mitotically inactive kinases retrieved from the public database. Also plant dis quiescent centre (QC), required to keep cells less differen ease resistance genes, which comprise a leucine-rich repeat tiated, is important to maintain the root meristem (van den and which are receptor-like were included in the alignment Berg et al. 1995; van den Berg et al. 1997). In the shoot (RPS5, RPS2, RPP8, RPM1, RPS4, N, RPP5, Meyers etal, meristem a balance between the WUSCHEL and CLAVATA 1999, Plant Ji, November20(3): 317-32). The multiple (CLV) genes has been implicated to play an essential role in alignment and phylogenetic tree of Said proteins were cal regulation cell division and differentiation (Clarket al. 1993; culated with the Clustal V method as described in example Fletcher et al. 1999; Schoof et al. 2000). The CLV1 gene 4. Because of the close proximity of RCH1 to CLV1 in the encodes a leucine rich repeat (LRR) receptor kinase (Clark, phylogenetic tree, there is more evolutionary relationship Williams, & Meyerowitz 1997), a member of a large gene between RCH1 and clavata, than with other kinds of LRR family (Lease, Ingham, & Walker 1998). Other members are receptor like proteins Such as plant disease resistance genes. ERECTA (ER) (Torii et al. 1996), BRASSINOLIDE INSENSITVE (BRI) (Li et al. 1997) and HAESA (HAE) 0311 FIG. 13. The nucleotide sequence of a minimal (Jinn et al. 2000), genes which have a function in plant promoter sequence of RCH1 development 0312 This figure shows the nucleotide sequence of SEQ 0321) We investigated whether root specific CLV1 ID NO. 18 that contains a minimal promoter sequence of the homologs exist using a differential RT-PCR based approach. RCH1 clone. This fragment of the genomic RCH1 clone is PCR products were generated from root tip and hypocotyl/ about 842 base pairs. This Sequence is deducted from the cotyledon cDNA pools using degenerate primers, resulting large promoter region Identified as SEQ ID NO 2 (see in the isolation of the root specific ROOT CLAVATA example 7). The minimal promoter region correlates with HOMOLOG 1 (RCH1). The promoter region of this gene the base pairs ranging from -1 of the first ATG defined in was translationally fused to B-glucoronidase-green fluores SEO ID NO. 2 to 842 of the first ATG defined in SEO ID cent protein (GUS/GFP) conferring root specific expression NO. 2. of these reporter genes (see Example 2). 0313 FIG. 14. Outprint of GenBank accession number AB017061 on Dec. 4, 2000. 0322 Designing Degenerate Primers 0323) To RT-PCR root specific members of the LRR 0314 FIG. 15. RCH1 prom::GUS expression in embryo's receptor kinase gene family we compared the Sequences of 0315 Arabidopsis embryo transformed with the CLV1, ER and HAE genes and their encoded proteins. RCH1 prom::GUS. Embryo's were stained with beta glucu The degenerate LRR primers were designed against the ronidase. The RCH1 promoter is active very early in plant NXLXGXIP encoding region of the XLXXNXLXGXIPXXLXX development and is only active in the root in the embryonic LXXLXXL consensus for the LRR receptor-like kinases. phase (A). On a stronger magnification (B) it is visible that Degenerate kinase primers were disigned for different the root Specific expression is limited to the meristematic homologous regions of the kinase domain. The primers are Zone of the embryonic root. listed in FIG. 2. 0316 FIG. 16. RCH1::GFP expression in roots 0324 RNA Isolation 0317 RCH1 promoter is strongly active in endodermis, 0325 For each RNA isolation the roottips and the hypo cortex, epidermis, lateral root cap, in short: in the “division cotyl/cotyledon part including the shoot apical meristem of Zone”. The RCH1 promoter is also active but in a low Arabidopsis thaliana (CoIO) plants, 4 days after germina manner, in the quiescent center and in the vascular tissue. tion, were collected. US 2004/OO67506 A1 Apr. 8, 2004 29
0326 Total RNA was isolated (Pawlowski et al. 1994) cDNA sample was amplified in a total volume of 50 ul as and chromosomal DNA contamination was removed upon described above for the differential RT-PCR. The RCH1 and Dnase I (Promega, Madison, Wis.) treatment, following the Ubi cDNAs were amplified during 26 or 20 cycles (30sec instructions of the Suppliers. The amount and quality of at 94° C., 30 sec at 56° C., 30 sec at 72° C), respectively RNA was determined using spectrophotometer and agarose (FIG. 4). RCH1 expression was only observed in root tip gel electrophoresis (data not shown). (R). 0327) Differential RT-PCR Example 2 0328 Reverse transcription (RT) was performed using 5 Promoter Isolation and Promoter Fusion Construct ug of root tip (R) and hypocotyl/cotyledon (C) total RNA in a 20 ul reaction volume with 0.5 lug oligodTs (Pharma 0337 To determine if the RCH1 promoter region is cia, N.J., USA) and 200 USuperscript II (LifeTechnologies, Sufficient to confer root Specific expression of a “gene of Paisley, UK), according to the instructions of the Supplier. interest' we decided to fuse this promoter to the Glucu Following first strand cDNA synthesis the cDNA samples ronidase/Green Fluorescent Protein (GUS/GFP) reporter gene. The RCH1 clone was used to isolate a phage contain were treated with RNase H (LifeTechnologies) as instructed ing the corresponding genomic region from a GEM by the Supplier and Subsequently diluted to a total Volume of (Promega, Madison, Wis.) based genomic library. The 100 ul with water. 2-GEM11 phage containing an insert of about 14550 nucle 0329 Amplification reactions were performed with all otides comprising the Arabidopsis thaliana RCH1 promoter degenerate LRR and KIN primer pairs (FIG. 2). For the and RCH1 gene and also the Surrounding genomic polymerase chain reaction (PCR) 2.5ul of the R or C clDNA sequences was, deposited on Oct. 25, 2000 at the BCCM Sample was amplified in a total volume of 50 ul reaction LMBP plasmid collection and was named “lambda phage volume containing 100 uM dNTPs, 100 ng LRR and KIN RCH1 genomic'. This deposit was given the accession primer, 1 U Taq polymerase (Boehringer, Ingelheim, Ger number LMBP 5582CB by the international depositary many) and its accompanying buffer. The cDNAS were authority. A restriction map was constructed of the genomic amplified during 40 cycles (1 min at 94 C., 1 min at 45-55 clone and a 7 kb XhoI fragment, containing 3.5 kb promoter C., 3 min at 72° C.). and part of the RCH1 coding Sequence, was Subcloned into 0330 Cloning and Sequencing the expression vector pBS (Stratagene, La Jolla, Calif.). The 3.5 kb promoter region including the first amino acids of the 0331. The amplified R and C cDNAs were separated and putative RCH1 ORF is set forth in FIG. 5 and is identified compared with agarose gel electrophoresis (FIG. 3). The as SEQID NO 2. This promoter region was amplified using root Specific fragments, e.g. fragments present in R22 and not the primers M13F and PRCH1 R and Pfu Taq polymerase in C, were isolated from gel, cloned into the pGEM-T (Stratagene, La Jolla, Calif.) in the manufacturer specified vector (Promega) and transformed into E. coli (strain conditions. The promoter region was Subsequently cloned DH5O), using Standard cloning procedures. into the binary pCAMBIA3383Xb vector (Roberts et al. 0332 Several PCR fragments of the same size but from 2000) creating a translational fusion with the GUS/GFP different members of the LRR receptor kinase gene family reporter gene. may be generated using degenerate primer pairs. Therefore, 0338 GUS histochemical Assay. 24 randomly picked colonies obtained after transformation were used in a “colony PCR'. Aliquots of the amplified 0339 Arabidopsis thaliana plants were transformed with inserts were separated on agarose gel followed by Southern this construct according to the vacuum transformation transfer to a Supercharge N+ membrane (Schleicher & method (Bechtold & Pelletier 1998). Transformants were Schuell, Dassel, Germany) to create a “colony blot'. An selected for phosphinothricin-tripeptide (PUT, Duchefa, aliquot of one insert was labeled with P-dCTP and hybrid NL) resistance. ized to the colony blot to determine the number of clones it 0340. Histochemical localization of B-Glucuronidase represented in the pool of 24. This procedure was repeated activity (GUS) was performed using the Substrate 5-bromo with other labeled inserts until all clones were identified. 4-chloro-3-indolyl glucuronide (X-gluc, BioSynth A G, 0333) A single representative of each pool of clones was Staad, Switzerland). X-gluc was dissolved in n-dimethyl Sequenced and the Sequence was analyzed using the BLAST formamide and diluted to 0.5 mg/ml in 50 mM NaPO4, pH program. One of the clones Sequenced was named RCH1 7.2 with 0.1% Triton X-100. Oxidative dimerization of the and showed Strong homology to plant LRR receptor kinases. produced indoxyl derivative was enhanced by adding the oxidation catalyst K" ferricyanide/ferrocyanide to a final 0334) RT-PCR concentration of 0.5 mM (0.5 mMK4Fe(CN).HO/0.5 mM 0335). The relative expression levels of RCH1 in root tip K3Fe(CN)). The GUS reaction was incubated at 37° C. (R), hypocotyl/cotyledon (C) and flowers (F) were deter over Several time periods, and pictures were taken from the mined using RT-PCR. Primers specific for the RCH1 cloured plant-tissues (FIGS. 7 and 15). In FIG. 7 the very sequence were designed (RCH1 F and RCH1 R, FIG. 2). Specific expression pattern of the RCH1 promoter is visual The PCR Samples were analyzed with agarose gel electro ized by the GUS reporter gene activity. Persons skilled in the phoresis. Ubiquitin (Ubi) mRNA was used as an internal art will also agree to the fact that the expression level of the control (Horvath et al. 1993). Polyubiquitingenes consist of RCH 1 promoter is very high, since after one hour the multiple units (Callis et al. 1995) and the Ubi primers Saturation level of Staining is reached in the meristem of the hybridize with the ends of each single unit. main root from a 1 week old seedling (FIG. 7D). 0336 Independently isolated total RNA was reverse tran 0341 RCH1::GFP expression in roots is shown in FIG. scribed to obtain cDNA and for the PCR reaction 2 ul of the 16. US 2004/OO67506 A1 Apr. 8, 2004 30
Example 3 in the art and outlined in the following paragraph. After Several time periods ranging from 1 day to 1 or more weeks, Alignment the seedling will be checked for the expression of the GUS 0342 Sequence alignment of one complete register reporter gene. This will be done by growing the Seedlings in Sequence against a target Sequence as in FIG. 10 was done organogenesis medium, and Staining them with glucu using the program GAP of the GCG package. The algorithm ronidase for Several time periods ranging from 1 hour to 5 of Needleman and Wunsch is applied here to find the hours. alignment of two complete Sequences (Needleman and Wun 0347 Agrobacterium-Mediated Rice Transformation sch, JMB 48(3): 443-453, 1970). The used parameters 0348. The Promoter-GUS hybrid gene outlined in during the alignment were Gap Weight: 8, Average Match: Example 2 can be transformed to Agrobacterium tumefa 2.912, Length Weight: 2, Average Mismatch: 2.003, Quality: ciens Strain LBA4404 or Css by means of electroporation 1388, Length: 1149, Ratio: 1.416, and Gaps: 25. and Subsequently transformed bacterial cells can be Selected Example 4 on a Solid agar medium containing the appropriate antibi otics. Phylogenetic Tree 0349 For demonstration of root-specific GUS expression with the promoter of the current invention, 309 mature dry 0343 For sequence comparison of proteins as in FIGS. Seeds of the rice japonica cultivars Nipponbare or Taipei are 11 and 12, the Clustal V method to align all the sequences dehusked, Sterilised and germinated on a medium containing was used (Higgins D G & Sharp PM, Comput Appl BioSci. 2,4-dichlorophenoxyacetic acid (2,4-D). After incubation in 1989 April;5(2):151-3). The Clustal Method groups the dark for four weeks, embryogenic, Scutellum-derived Sequences into clusters by examining Sequence distances calli are excised and propagated on the Same medium. between all pairs. Clusters are aligned as pairs then collec Selected embryogenic callus is then co-cultivated with tively as Sequence groups to produce the overall alignment. Agrobacterium. Co-cultivated callus is grown on 2,4-D- After the multiple alignment is completed, a neighbor containing medium for 4 to 5 weeks in the dark in the joining method is employed to reconstruct phylogeny for the presence of a Suitable concentration of the appropriate putative alignment. Selective agent. During this period, rapidly growing resistant 0344) The Clustal method uses weight tables to construct callus islands develop. After transfer of this material to a multiple alignments. Residue weight tables are used in medium with a reduced concentration of 2,4-D and incuba Scoring protein and nucleotide alignments So that slightly tion in the light, the embryogenic potential is released and mismatched residues, Such as lie VS. Val amino acids or GVS. shoots develop in the next four to five weeks. Shoots are R nucleotides, are not Scored the Same as total mismatches. excised from the callus and incubated for one week on an For the alignment and pylogenetic trees in FIGS. 11 and 12 auxin-containing medium from which they can be trans the PAM250 weight table was used. Other method param ferred to the Soil. Hardened shoots are grown under high eters for pairwise alignment were K-tuple: 1, Gap Penalty: humidity and short days in a phytotron. Seeds can be 3, Window: 5 and Diagonals: 5. Other method parameters harvested three to five months after transplanting. The for multiple alignment include Gap Penalty: 10 and Gap method yields Single locus transformants at a rate of over Length Penalty: 10. 50% (Chan et al. 1993; Hiei et al. 1994) 0345 To view phylogenetic relationships compatible Example 6 with multiple Sequence alignments a Phylogenetic Tree was calculated. The length of each pair of branches represents Expression of a Cytokinine Oxidase cDNA Under the distance between Sequence pairs, while the units at the the Root-Specific RCH1 Promoter in Transgenic bottom of the tree indicate the number of Substitution events. Tobacco and Rice Leads to increased Root The phylogenetic tree gives a first estimate of the relation Production ship between homologous Sequences. A cladogram presen 0350. The AtCKX1 gene (located in the database with the tation of the phylogenetic tree is shown in FIGS. 11 and 12. accession number: AC002510, Arabidosis thaliana chromo Some II section 225 of 255 of the complete sequence, Example 5 sequence from clone T32G6) can be cloned under the control of the RCH1 promoter of Arabidopsis, in a binary Root-Specific Report r Gene Expression in Rice vector. Also the ZmCKX1 from maize (accession number Mediated by the Arabidopsis thaliana RCH 1 AF044603, (Morris et al. 1999) can be operably linked to the Promoter RCH1 promoter a binary vector. These genes can be trans 0346) CLAVATA homologs are found in many other plant fected into Tobacco or Rice plants, using agrobacterium Species from both the groups of monocots and dicots. mediated gene transfer. The transgenic plants expressing the Therefore one can assume that also the promoter of RCH1 ATCKX1 or the ZmCKX1 specifically in roots are expected is functional in those other plant species, and possibly exerts to show increased root production without negatively affect the same Specific expression-pattern. To investigate the ing shoot development and without inducing premature leaf feasibility of root meristem-specific activation of the Ara SCCSCCCC. bidopsis thaliana RCH1 promoter in monocots, the afore Example 7 mentioned promoter-reporter gene construct, wherein the GUS/GFP chimeric gene is operably linked to the RCH1 Minimal Promoter Sequence promoter, will be transformed to rice using the Standard 0351) The length of the RCH1 promoter region (SEQ ID transformation procedures well known to the perSons skilled NO2), which confers to root specific expression patterns, US 2004/OO67506 A1 Apr. 8, 2004
can be reduced to a "minimal promoter' without altering the sequences (AB011476-pro, T05050 pro and AC015446.pro) root Specific expression patterns or other features of the are used in knockout experiments or in antisens expression. promoter Such as constitutive and Strong activity. AS an example, the length of the promoter region was reduced to 0357) Ectopic Expression of RCH1 a minimal promoter Sequence ranging from the -1 base pair 0358. The expression of RCH1 in normal cells correlates just before the start codon at position 3479-3481 of SEQ ID with the Zone of rapidly dividing cells S and is involved in NO. 2 to -842 base pairs before said start codon. Said the maintenance of meristem cells and dividing cells. minimal promoter is represented in SEQ ID NO. 18. This Improved activity of RCH1 in a host cell results in the minimal promoter was PCR amplified and translationally maintenance of cell division in that host cell and thus works fused to the GUS reporter gene. With a GUS histochemical against the differentiation of that cell. On the contrary assay (example 2) the same expression pattern as the large diminished RCH1 activity in a host cell results in a dimin promoter region was demonstrated (results not shown). The ished cell division in that host cell and thus in the loss of the difference is that the sequence before the HindIII site at the amount of dividing cells in the meristem. Particular beginning of the minimal promoter is not necessary for the examples to influence these processes by altering the expres root Specific expression pattern. sion level of the RCH1 gene are described in the following paragraphs: Example 8 0359. Overexpression RCH1 prom::GUS Expression in Embryo's 0360. In order to obtain higher activity of the RCH1 0352 Siliques of Arabidopsis plants transformed with protein in the host cell the coding region of the RCH1 gene RCH1 prom::GUS were opened at different stages of devel is fused to a strong promoters that is inducible or constitutive opment and the embryo's were Surgically taken out of the and/or ubiquitous or tissue specific. ovules and transferred to X-gluc Staining Solution. Embryo's 0361) To influence the RCH1 activity in a particular set of were stained for 0.5h-4h before pictures were taken using host cells, the RCH1 coding region is cloned under control light microscopy, FIGS. 15A and B are at 25x and 40x of other meristemic promoterS Such as the promoters of magnification respectively, Stained for 0.5 h. Longer Staining WUSCHEL, CLV1, CLV2, CLV3, STM. The obtained effect did not reveal expression outside the root promeristem is the control of the expression of RCH1 in a particular area region. of the meristem or in a particular time period of the 0353 RCH1 is active very early: RCH1 expression was development of the plant. Also the promoters of other RCH first observed in torpedo Stage embryo's in the root prom genes (such as AB028621.pro, AB011476-pro, TO5050 pro eristem and was maintained in this region during the later and AC015446-pro) are used for this purpose. Stages of development. This figure shows that as Soon as a 0362. In a particular application of the present invention, meristem is formed RCH1 is expressed and provides further the RCH1 gene and the RKN gene (=the rice homologue of evidence that RCH1 expression is correlated with the RCH1 as described in WO/04761) are operationally linked appearance and presence of a root meristem from the embry to the root-specific promoter PYK10, a constitutive ubiq onic Stage onwards. uitin or GOS 2 promoter and Cdc2a promoter and expressed in Arabidopsis or in rice. Example 9 0363 Downregulation Isolation of cDNA of Other Arabidopsis RCH1 0364) In order to obtain lower activity of the RCH1 Homologues protein in the host cell, the coding region of the RCH1 gene 0354) cloned in a Sense and antisense orientation in order to form a hairpin that induces gene-Silencing in the host cell. TABLE 4 0365 Co-Expression % identity with RCH1 0366 Downregulation or upregulation of the RCH1 in % sequence combination with downregulation or upregulation of other Database entry Database entry identity with geneS e.g.: genomic DNA protein RCH1 on aa level RCH1.2 ABO28621 - APOO2O37 BABO3O91 59.8% 0367. With WUSHEL: RCH1.3 ABO11476 BABO9286 40.1% RCH14 ALO22223 CAA1826 or 38.9% 0368. In the shoot meristem a balance between the WUS TO5050 CHEL and CLAVATA (CLV) genes has been implicated to RCH1.5 ACO154.46 AAG12526 42.1% play an essential role in regulation cell division and differ entiation (Clark et al. 1993; Fletcher et al. 1999; Schoof et al. 2000). The whole meristem maintenance is controlled by 0355 To discover the exact function of RCH1, the inven these genes and they seem to have an opposite function tors want to circumvent the redundancy of the gene. keeping the balance between cell division and differentia tion. Therefore, WUSCHEL or a root WUSCHEL homo 0356 AB028621 (see FIG. 12, phylogenetic tree) is logue is a preferred partner to be co-expressed with RCH1. determined to be RCH1.2 protein and is also used by the inventors for further experiments. Double knock outs of 0369. In an application of the invention RCH1 is upregu RCH1 and RCH1.2 were made and also other closely related lated in combination with the downregulation of WUS US 2004/OO67506 A1 Apr. 8, 2004 32
CHEL in a root specific manner. The obtained effect of this 0380 Banerjee A. et al. (1996) Omega amino acids in co-expression is a better control on the meristem mainte peptide design: incorporation into helices. Biopolymers CC. 39, 769-777. 0370. With Other RCH1 Family Members 0381 Baron M. H. & Baltimore D. (1982) Antibodies 0371 Possibly the function of RCH1 is redundant and against the chemically Synthesized genome-linked pro other closely related genes can take over its function. tein of poliovirus react with native virus-specific pro Therefore the RCH1 gene ectopically expressed in combi teins. Cell 28, 395-404. nation with closely related genes in order to downregulate or 0382 Bartel, P. L. & Fields, S. (1997) The Yeast upregulate the activity of the encoded protein. The RCH1 Two-Hybrid System, Oxford University Press. homologues used for co-expression are for example 0383 Baszczynski, C. L., Fallis, L., Bellemare, G., and AB028621.pro, AB011476.pro, TO5050 pro and AC015446 Boivin, R. Brassica regulatory Sequence for root-spe (see FIG. 12 and table 4). The obtained effect is a better cific or root-abundant gene expression. WO9007001. effect as described for the ectopic expression of RCH1 2OOO. alone. 0384 Bechtold N. & Pelletier G. (1998) In planta 0372 With Other Clavata Homologues: Agrobacterium-mediated transformation of adult Ara 0373 Clavata 1 is a receptor that is involved in a signal bidopsis thaliana plants by vacuum infiltration. Meth pathway probably through the binding of a ligand (e.g. Ods Mol. Biol. 82, 259-266. Clavata 3). A particular useful application of the present invention is the co-expression of RCH1 with the root 0385 Bell M. H. et al. (1993) Tobacco plants trans Clavata 3 homologue RCH3. The obtained effect is a better formed with cdc25, a mitotic inducer gene from fission control on meristem maintenance. yeast. Plant Mol. Biol 23, 445-451. 0386 Benkirane N. et al. (1996) Exploration of Example 10 requirements for peptidomimetic immune recognition. Antigenic and immunogenic properties of reduced pep Domain Swapping tide bond pseudopeptide analogues of a histone 0374. The N-terminal LRR domain of RCH1 functions as hexapeptide. J. Biol. Chem. 271, 33218-33224. an extracellular perception domain and a C-terminal kinase 0387 Berry A. & Brenner S. E. (1994) A prototype domain functions in an intracellular signal transduction computer System for de novo protein design. Biochem. pathway. Previously it was described by He et al. (Science Soc. Trans. 22, 1033-1036. 2000, 288(5475): 2360-3: Perception of brassinosteroids by the extracellular domain of the receptor kinase BRI1) that 0388 Binarova P. et al. (1998) Treatment of Vicia faba Such domains can be Swapped with the domains of other root tip cells with Specific inhibitors to cyclin-depen proteins. Following this methodology in a particular appli dent kinases leads to abnormal Spindle formation. Plant cation of the present invention, the domains of RCH1 are J. 16, 697–707. Swapped with the domains of another protein. In one 0389 Bögre L. et al. (1997) The cdc2Ms kinase is example the LRR domain of RCH1 is replaced by the LRR differentially regulated in the cytoplasm and in the domain of BRI. The effect obtained is the activation of a nucleus. Plant Physiol. 113, 841-852. different pathway, which is normally not activated by RCH1, because the kinase domain of RCH1 now responds to 0390 Bögre L. et al. (1999) AMAP kinase is activated another external Signal that normally is not recognized by late in plant mitosis and becomes localized to the plane RCH1. of cell division. Plant Cell 11, 101-113. 0375. It will be clear that the invention may be practiced 0391) Borg S. et al. 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SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS : 19
<210> SEQ ID NO 1 &2 11s LENGTH 74.38 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana US 2004/OO67506 A1 Apr. 8, 2004 38
-continued <400 SEQUENCE: 1 citcgagtttc agatgtttct attaaataag taatgaaag.c agctotaatt citcccaaaat 60 to acaagtat agaaagatag aaagatagaa acagagittat to catggaag citgaaaatca 120 tacago atgg toggatataac caatgaga.ca caaaacaatc acgg catgttg ttatagaa.ca 18O acacttatca totgtattaa cataaagcaa aagttccitat acg acatagt coaaagttac 240 agaccataag cacaccittat gatttgttgt accctictaaa acacaggatg atgacaagaa 3OO gcaaatttgc ccaaatttac to agggaaaa acgagggctt aacgctotca tat citcaaac 360 aaaaacttct caaacagttt catgttgttca gattggaggg caatggcaac ggataagctg 420 ccatcattag tagg acttgg tag cacaaaa gacaaac colt c.gitatggaat tocaccaggt 480 cc cataaaga taggacg acc ccaac caaag totgcatcat aaataggtaa totalacc cag 540 cittgttgattc ccaaatttgg gcacttgtag gtatgttgcac cqcggacaag ggctgacaga 600 to aggctgca totcCaggta gtcaagagct gacct cagat agittatcatc catacga acc 660 aagaaatcat gaatctgtcc tdcagcatac catgttggct tagataacaa atc.ccctgca 720 acagocaatg gtgttgcagt gaatatocaca titc.ccaaagt aaccaggagg cagotgcgga 78O cgtagt ctag accittc catc agttgcaatg tacagtttcg totcittgg to gtttggaagc 840 cctogcgcct titcccactga totccacaca tag.ccctgcca acatctogta tdag citgtag 9 OO citgacagtgt toccatcc to cittggattitc gccittaa gag caacaagct g g to tcgtgtt 96.O aatttgaata tagagacagt gg tattotca ggtoctoatt tag acggatc aagaggitatc O2O ttcatacttg gtgcaggctd atattoaiaca tatgaaaag caggctgtgg cqggtoccita O8O gctcggagga gtgttc gatc aatgaaaggt ggaatggitta ggtoaag acc acgagccata 14 O to agaic catg tdttgataaa atgaagacca gagaalaccat citgcc.gc.gtg atgttgcatc 200 ccaaccocaa gtgaagctcc cccacattta aagaaagttca cotatatoat caaacaatat 260 cattacataa citaaaaacag alacatacatc atcaaagaat atcattacag alactaaaaaa 320 cagaacaa.ca caag cattca ataaattaaa aaagggaaag actittaaatt aagct gacct 38O gcaaaacgag aag.cgggaaa gagtgaatgc cagcggagtg atcaactitc g g gaataagct 4 40 gacggagatt aagggtagga gcaaaatcac caaaatcatc gatalacagaa gagtaticag 5 OO ccacaacgaa gagaacacca gcaccgttac aatcgatcto aatac gacca to atcgtctic 560 tottcaag.cg accago cata gggtaaaaag ggacaagggc tittggaaaga gct tcc titca 62O tgacct gagg gtcaaagaaa ttggaag.cgc cqgtgggtot gtagaagtag acact agggg 680 tatggaatct g g g gatgaca aggtogacgt toggagttcca aagattagt g attggtgtct 740 cggtgg cagg ccggaccatg gtggaatcto tdatgttaat titt catattt ttaagaaaga 800 toaaacgaac aaacaaattt tottttgagt ttgatctgag aaattgttgttg agaag.cgagg 860 aggggtag at ataagtgggt gtgaacaaaa aaggaaaggit cqtaatcctt caccitaccgc 920 cccacg acco cqcctactcc atctgtcgac caactictotc agaaggttta toattggtoc 98O atctoatcac toggtoctac titttctatoc g g g gttggta aaccogttcc citctgtc.cta 20 40 aggcc.caaat aatactaatg g g cittaaata gtataaagtt cqaatgaaga agaaggaaaa 2100 aaaattgaca agttgattgt tatagotacic gtacaagtta gtcaatc gala toccaaagtt 216 O tggcttaact taactcatta ccacgtag ca gag catalaca acaaccacca ataa.catcta 2220 US 2004/OO67506 A1 Apr. 8, 2004 39
-continued acgg.cgtaaa citcacgtgtt aagagaatta cqtcagaatc caagaaagtic tittttitt cag 228O tacaccacaa ataaattatgttgaattitat atcc.gaacta tactatatta tatgaaaaca 234. O tacctdaatt agtcataaat titcacatttc attittaattt attaactaat g gttgttgtt 24 OO gcggatagot tittctittgct ttctgttcta aaattatttc ttagccatta tittgatgatc 2460 caacttggitt to aggatcta ataaaaattic aatttctittg aag cattcat tiggcttaatg 252O atccaaacct tttgtc.citcc cactcitctaa gaaaataaag caaaagcaaa actaaaagaa 258O aa.gcaactag taccgtgggit togtocaatgg talacc catgc taccaaatac caatcaaag.c 264 O ttaaatgaca atttagtacc ttgggttggit catgatttag agcggaacaa atataccata 27 OO catcaaacga ggatatacag agaaaattica toggaagtatg gaatttagag gacaatttct 276 O. cittctgggct acaacgg acc gg.cccattcg citcatttacc cagaggitatic gagtttgttgg 282O acttittgatg cc.gctagaga citattggcat cqgattgaaa aaaatgttta citt.cgttgtt 2880 aacaattittctgaatgcaat atttitccttg tdatgaatat ttaaacttgt tattactittc 2.940 ttittagctta ggtgtggaca attatggagt titactitcaaa cigaggaagaa tottaaacgc 3OOO toggttcagg totcgaaaac aaaccaactc acaatcc to a cittaattgag gaaaacaatg 3060 caaaaccaca to catgctitc catatttcta toataatctt ataagaaaaa acactactaa 312 O gtgaaatgat tctgtatata tataaccaat gccttttgtt ttgttgatatt titatgtatat 318O ataactattg actitttgtca totatggata gtgtctoggg citcttggcaa acatatttca 324 O aagaaaagtt aatgactgta attaattaat citgaagctag aaacagaacc cc.gaggtaaa 3300 agaaaaag acagag cacatg aagtttagta cittittatata tittaatatat cattctttct 3360 tattgctitat citctaaag.ca aaaactitc.cc taalacccitaa gocaaaggac toagatc gat 342O gcagaaccala gaaggcttgt tittggatttg agagccaaat gcaaagaaaa aaact cittat 3480 gtotttgcaa atgccg attc caagaaaaaa agccittaact gtttctoatt tittccattac 354. O attgtctotg tttittagctt tottcatcto citcg accitct gcatcaacca atgaagttcto 3600 agctittgatt tottggctitc acagotcgaa citc.gcc.gc.ca cc.gtoagtot totcc.ggctg 3660 gaatccttct gattctgacc catgtcaatg gccttacatt acttgttcct cotcagacaa 372 O caaactcgtt acagagatca atgtcgtotc tagttcagtta gctttacctt toccitcctaa 378 O. catttctitcg tttactitcac titcagaaact cqttatctoc aacactaatc. tcaccggagc 384 O tatatottct gagatcggag attgttcgga gcttatagitt atcgatttaa gotcaaatag 39 OO tottgttggit gaaatacctt cqagtictagg gaagctcaag aatcttcaag aactttgttt 396 O aaactccaat ggc citcacag gaaagatc.cc accggaacto gg.cgactgog ttagcct caa 4020 gaatcttgag attittcgata act acttatc cq agaatcto cogttggagc ticggaaagat 408 O citcg actott gaga.gcataa gag coggagg aaacticagag citttcaggga agatc.ccgga 414 O ggagat.cgga aactgtagga atctoaaggit cittagggitta gcago aacga aaatato cqg 4200 ttctttacct gtttcgttgg gtoaactaag caagctocag totttatctg. tctattotac 4260 aatgct citca ggtgagatcc ctaaagagct toggaaactgc tict galactta totaatctgtt 4320 totatacgac aatgacittgt ccggcacgct tccaaaagaa citaggaaagc titcaaaacct 4.380 agagaagatg cittctato go agaataatct coacggacct atc.cctgagg agattggatt 4 440 catgaaaag.c ttaaacgc.ca ttgatctotc tatgaactat ttctoaggaa ccatccctaa 4500 US 2004/OO67506 A1 Apr. 8, 2004 40
-continued atcgttcggit aacttgtcga atctocaaga gct tatgctt agcagdalaca acatcaccgg 45 60 gtocaatacct tcgattictaa gtaactgcac aaagcttgtt cagttccaga ttgacgc.cala 462O toagatttica ggtttgatto caccagagat toggattgctt aaggagctoa acatcttctt 4680 gggatggcag aataagctag aagggaat at toc ggacgag titagctggitt gttcagaatct 474. O tdaag citctt gacittgtcac agaattatct caccggatca ttacctgcag gtttgtttca 4800 gctitcgtaat citg actaagc tottgctitat atctaatgcc atctocqgtg ttatcccact 4860 ggagat.cggg aactgcactt cqcttgtcag attaaggctt gtcaataa.ca gaatcaccgg 4920 agaaatcc ct aaagg tattg gatttcttica galaccittagc titccitcqact totctgagaa 4.980 caatctotcc ggtocagttc ctittggagat aagtaattgt agacaacto c agatgcttaa 5040 cittgagcaac aac actictitc aaggittatct tccitctgtct ttatcatcgt taacaaagct 51OO tdaagtactt gatgtc.tc.tt coaatgacitt gactggcaag atacctgata gtc.tagg to a 5 160 totcatttca citcaaccq go toattctaag taagaattct ttcaatggag aaatccctitc 5220 gtotctoggt cactgcacga atcttcagot tottgatcto agcagtaata acatctotgg 528 O aactataccg gaggagcttt ttgacatcca agatctagac attgc gttaa acttgagctg 5340 gaattic atta gatggctitta toccggaaag gattitcc.gc.g. cittaa.ccgat tatcc.gtgct 5 400 cgatatttcg catalacatgc titt caggcga cctttctg.cg citgtc.cgg to tagaaaactt 546 O ggtttctotg aacatctotc ataacagatt citcaggittat cittccagata gitaaagttgtt 552O tag acagotgataggagcag agatggaagg aaacaatgga citctgttcca aaggitttcag 5580 gtottgctitt gtaagtaa.ca gttcacagtt alactacacag cqtggtgtgc acticacatag 5640 actcaggata gcc attggat tdctaatcag cqtgacagog gttctagogg tattagg.cgt. 5700 gttggcggitt atacgagcta aacaaatgat to gagatgat aac gatticag agacitggaga 576 O. aaatctatog acatggcaat to acaccittt to agaaacto aactt cacag togalacatgt 582O actcaagtgt ttggtagaag gtaatgttat agggaaaggit togcto cqgga tagtgtacaa 588 O agctgaaatg cctaacagag aagttcatcgc ggtgaaaaag citctggc.cag togacagtacc 594 O taatctgaat gagaaaacta agt catcagg agttc gagac to attcticag citgaagtaaa 6 OOO aacacttgga to gatcagac acaagaac at totacgctitc ttgggatgtt gttggaacaa 6060 galacactaga cittctitatgt atgattatat gtcaaatggg agtttgggaa gtttgcttca 61.20 cgaaaggagc ggtgitatgta gcttaggatg g galagtogagg tacaagatta tacttggtoc 618O agcticagggit ttggct tact to caccatga citgtgttcct cocattgttc atagaga cat 624 O caaggcaaat aatattotga ttggccct ga ttittgaacct tacattggag attitcgg act 6300 cgctaagctt gttgatgatg gcg actittgc ticgttctitcc aacac cattg citggttccta 6360 tggittacata gctccaggta cittcc caaat ttctgttittg citgctaatta gttaacttga 642O tacatcataa gttact agta gtc.ttagoct agtaatggta gaaattcagg tittatagttt 64.80 ggittittggitt tittggcattt togaaaagaat agtattittgg titcagttgat titt.cgatttg 654. O gtttaaaatt ttagaaaaaa aatggtag aa gaaataattt ttgattaata ttatato atc 6600 tact.gtatac gatttaaaaa aaaaattgat ttggitttggit citcgatattt tacttgtagc 6660 ttggttcaat atttittacta tatatatgat tcatgactitt catactaatc aatatttgtt 672O cggitttcgtt ttgattagtc. tcaaagttgaa toatatagitt aaaatattga ccaatctato 678 O. US 2004/OO67506 A1 Apr. 8, 2004 41
-continued atatacttac acgctacaag atttitcgatg taattittggit gtaag cattt tdgtttittat 6840 atgttcatcc ttagtaacta acaacaaata totaattggga agcataacgt tatatattoc 69 OO cataaggcta talacactata attatgcaga atacggatac toaatgaaga talacagagaa 696 O aag.cgacgtg tacago tatg gag togtagt gctic gaggta citaacgggta agcaaccaat 7 O2O cgatccaacg ataccagatg gactic cacat agtgg actogg gtcaagaaaa totagaga cat 708O acaagtaatc gaccaaggac tacaa.gcaag accagagtca gaggttgaag agatgatgca 714. O aacgctagga gtc.gc.gcttt tatgcattaa tocaatacco galagacaggc ctacaatgaa 72OO agatgtggct gctatoctita gtgagatatgtcaagagaga gaggaatcga tigaaagttga 726 O tggttgctcg ggaagttgta acaatggaag agaacgtggc aaagatgatt cqactitcatc 732O ggittatgcaa caaacggcta agtatttgag aagtagtagc acgagtttct citgcgtottc 738O tittgctttac tottcttctt cittctgctac ttctaatgtt agaccaaatc ttaaataa 74.38
<210> SEQ ID NO 2 &2 11s LENGTH 34.99 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 2 citcgagtttc agatgtttct attaaataag taatgaaag.c agctotaatt citcccaaaat 60 to acaagtat agaaagatag aaagatagaa acagagittat to catggaag citgaaaatca 120 tacagoatgg toggatataac caatgagaca caaaacaatc acgg catgttgttatagaa.ca 18O acacttatca totgtattaa cataaagcaa aagttccitat acg acatagt coaaagttac 240 agaccataag cacaccittat gatttgttgt accctictaaa acacaggatg atgacaagaa 3OO gcaaatttgc ccaaatttac to agggaaaa acgagggctt aacgctotca tat citcaaac 360 aaaaacttct caaacagttt catgttgttca gattggaggg caatggcaac ggataagctg 420 ccatcattag tagg acttgg tag cacaaaa gacaaac colt c.gitatggaat tocaccaggt 480 cc cataaaga taggacg acc ccaac caaag totgcatcat aaataggtaa totalacc cag 540 cittgttgattc ccaaatttgg gcacttgtag gtatgttgcac cqcggacaag ggctgacaga 600 to aggctgca totcCaggta gtcaagagct gacct cagat agittatcatc catacga acc 660 aagaaatcat gaatctgtcc tdcagcatac catgttggct tagataacaa atc.ccctgca 720 acagocaatg gtgttgcagt gaatatocaca titc.ccaaagt aaccaggagg cagotgcgga 78O cgtagt ctag accittc catc agttgcaatg tacagtttcg totcittgg to gtttggaagc 840 cctogcgcct titcccactga totccacaca tag.ccctgcca acatctogta tdag citgtag 9 OO citgacagtgt toccatcc to cittggattitc gccittaa gag caacaagct g g to tcgtgtt 96.O aatttgaata tagaga cagt gg tattotca ggtcc to att tag acggat.c aagaggitatic 1020 ttcatacttg gtgcaggctd atattoaiaca tatgaaaag caggctgtgg cqggtoccita 1080 gctcggagga gtgttc gatc aatgaaaggt ggaatggitta ggtoaag acc acgagccata 1140 to agaic catg tdttgataaa atgaagacca gagaalaccat citgcc.gc.gtg atgttgcatc 1200 ccaaccocaa gtgaagctcc cccacattta aagaaagttca cotatatoat caaacaatat 1260 cattacataa citaaaaacag alacatacatc atcaaagaat atcattacag alactaaaaaa 1320 cagaacaa.ca caag cattca ataaattaaa aaagggaaag actittaaatt aagct gacct 1380 US 2004/OO67506 A1 Apr. 8, 2004 42
-continued gcaaaacgag aag.cgggaaa gagtgaatgc cagcggagtg atcaactitc g g gaataagct 4 40 gacggagatt aagggtagga gcaaaatcac caaaatcatc gatalacagaa gagtaticag 5 OO ccacaacgaa gagaacacca gcaccgttac aatcgatcto aatac gacca to atcgtctic 560 tottcaag.cg accago cata gggtaaaaag ggacaagggc tittggaaaga gct tcc titca 62O tgacct gagg gtcaaagaaa ttggaag.cgc cqgtgggtot gtagaagtag acact agggg 680 tatggaatct g g g gatgaca aggtogacgt toggagttcca aagattagt g attggtgtct 740 cggtgg cagg ccggaccatg gtggaatcto tdatgttaat titt catattt ttaagaaaga 800 toaaacgaac aaacaaattt tottttgagt ttgatctgag aaattgttgttg agaag.cgagg 860 aggggtag at ataagtgggt gtgaacaaaa aaggaaaggit cqtaatcctt caccitaccgc 920 cccacg acco cqcctactcc atctgtcgac caactictotc agaaggttta toattggtoc 98O atctoatcac toggtoctac titttctatoc g g g gttggta aaccogttcc citctgtc.cta 20 40 aggcc.caaat aatactaatg g g cittaaata gtataaagtt cqaatgaaga agaaggaaaa 2100 aaaattgaca agttgattgt tatagotacic gtacaagtta gtcaatc gala toccaaagtt 216 O tggcttaact taactcatta ccacgtag ca gag catalaca acaaccacca ataa.catcta 2220 acgg.cgtaaa citcacgtgtt aagagaatta cqtcagaatc caagaaagtic tittttitt cag 228O tacaccacaa ataaattatgttgaattitat atcc.gaacta tactatatta tatgaaaaca 234. O tacctdaatt agtcataaat titcacatttc attittaattt attaactaat g gttgttgtt 24 OO gcggatagot tittctittgct ttctgttcta aaattattitc titagccatta tittgatgatc 2460 caacttggitt to aggatcta ataaaaattic aatttctittg aag cattcat tiggcttaatg 252O atccaaacct tttgtc.citcc cactcitctaa gaaaataaag caaaagcaaa actaaaagaa 258O aa.gcaactag taccgtgggit togtocaatgg talacc catgc taccaaatac caatcaaag.c 264 O ttaaatgaca atttagtacc ttgggttggit catgatttag agcggaacaa atataccata 27 OO catcaaacga ggatatacag agaaaattica toggaagtatg gaatttagag gacaatttct 276 O. cittctgggct acaacgg acc gg.cccattcg citcatttacc cagaggitatic gagtttgttgg 282O acttittgatg cc.gctagaga citattggcat cqgattgaaa aaaatgttta citt.cgttgtt 2880 aacaattittctgaatgcaat atttitccttg tdatgaatat ttaaacttgt tattactittc 2.940 ttittagctta ggtgtggaca attatggagt titactitcaaa cigaggaagaa tottaaacgc 3OOO toggttcagg totcgaaaac aaaccaactc acaatcc to a cittaattgag gaaaacaatg 3060 caaaaccaca to catgctitc catatttcta toataatctt ataagaaaaa acactactaa 312 O gtgaaatgat tctgtatata tataaccaat gccttttgtt ttgttgatatt titatgtatat 318O ataactattg actitttgtca totatggata gtgtctoggg citcttggcaa acatatttca 324 O aagaaaagtt aatgactgta attaattaat citgaagctag aaacagaacc cc gaggtaaa 33OO agaaaaag acagag cacatg aagtttagta cittittatata tittaatatat cattctttct 3360 tattgctitat citctaaag.ca aaaactitc.cc taalacccitaa gocaaaggac toagatc gat 342O gcagaaccala gaaggcttgt tittggatttg agagccaaat gcaaagaaaa aaact cittat 3480 gtotttgcaa atgcc.gatt 34.99
<210> SEQ ID NO 3 &2 11s LENGTH 3960 &212> TYPE DNA US 2004/OO67506 A1 Apr. 8, 2004 43
-continued <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 3 atgtctittgc aaatgccg at tocaagaaaa aaag.ccttaa citgtttctoa tttittccatt 60 acattgttcto tgtttittagc tittcttcatc. tccitcg acct citgcatcaac caatgaagttc 120 tdagctittga tittcttggct tcacagotcg aactc.gcc.gc caccgtoagt cittctocq go 18O tggaatccitt citgattctga cccatgtcaa tagg ccttaca ttacttgttc citcctcagac 240 aacaaactcg ttacagagat caatgtcg to tctgttcagt tagctttacc tittcc citcct 3OO aacatttctt cqtttacttic actitcagaaa citcgittatct coaacactaa totcaccgga 360 gctatatott citgagatcgg agattgttcg gag cittatag titatcgattit aag citcaaat 420 agtc.ttgttg gtgaaatacc titcgagtcta gggaagctoa agaatcttca agaactttgt 480 ttaaactcca atggccitcac aggaaagatc ccaccggaac toggcgact g c gttagcctic 540 aagaatcttg agattittcga taact actta toc gagaatc. tcc.cgttgga gctcqgaaag 600 atctogactic titgagag cat aagagcc.gga ggaaacticag agctttcagg galagatc.ccg. 660 gaggagat.cg gaaact gtag gaatctoaag gtc.ttagggit tag cagdaac gaaaatato c 720 ggttctttac citgtttcgitt g g g toaacta agcaagctoc agtctittatc tdtctattot 78O acaatgctict caggtgagat coctaaagag cittggaaact gctctgaact tat caatctg 840 tittctatacg acaatgacitt gtc.cggcacg citt.ccaaaag alactaggaaa gottcaaaac 9 OO citagagaaga togcttctatg gcagaataat citccacggac citatccctgaggagattgga 96.O ttcatgaaaa gottaaacgc cattgatcto tctatgaact atttcto agg aaccatccct O20 aaatcgttcg gtaacttgtc. gaatctocaa gag cittatgc titagcagdala caa.catcacc O8O gggtocaatac citt.cgattct aagta actgc acaaagcttg titcagttcca gattgacgc.c 14 O aatcagattt caggtttgat tocaccagag attggattgc titaaggagct caa.catcttic 200 ttgg gatggc agaataagct agaagggaat attcc.ggacg agittagotgg ttgtcagaat 260 cittcaagcto ttgacttgtc. acagaattat citcaccqgat cattacctg.c aggtttgttt 320 cagottcgta atctgactaa gotcttgctt atatotaatg ccatctocqg tdttatccca 38O citggagat.cg ggaactgcac titcgcttgtc. agattaaggc titgtcaataa cagaatcacc 4 40 ggagaaatcc ctaaaggitat toggatttctt cagaaccitta gctitcctic ga cittgttctgag 5 OO aacaatctot cogg to cagt toctittggag ataagtaatt gtag acaact coagatgctt 560 aacttgagca acaacactct tcaaggittat cittccitctgt ctittatcatc gittaacaaag 62O cittcaagtac ttgatgtc.tc titccalatgac ttgactogca agatacctga tag to taggit 680 catctoattt cactcaaccq got cattcta agtaagaatt citttcaatgg agaaatccct 740 togtotctog gtcactgcac gaatcttcag cittcttgatc tdag cagtaa taacatctot 800 ggaactatac cqgaggagct ttittgacatc caagatctag acattgc gtt aaacttgagc 860 tgga attcat tagatggctt tatcc.cggaa aggattitcc g c gottaa.ccg attatcc.gtg 920 citcgatattt cqcatalacat gctitt caggc gacctttctg. c.gctdtc.cgg totagaaaac 98O ttggitttcto tgaacatcto tcataacaga ttctdaggitt atctitccaga tagtaaagtg 20 40 tittaga cago toataggagc agagatggaa gqaaacaatg gacitctgttc caaaggtttc 2100 aggtottgct ttgtaagtaa cagttcacag ttalactacac agcgtggtgt gcacticacat 216 O US 2004/OO67506 A1 Apr. 8, 2004 44
-continued agacitcagga tagccattgg attgctaatc agcgtgacag cq gttctago ggit attaggc 2220 gtgttggcgg ttatacgagc taaacaaatg attc.gagatg ataac gattic agagacitgga 228O gaaaatctat ggacatggca attcacacct titt cagaaac toaacttcac agtcgaacat 234. O gtactcaagt gtttggtaga aggtaatgtt atagg gaaag gttgcto cqg gatagtgtac 24 OO aaagctgaaa toccita acag agaagttcatc gcggtgaaaa agctotggcc agtgacagta 2460 cctaatctgaatgagaaaac taagttcatca ggagttc gag act cattct c agctgaagta 252O aaaacacttg gatcgatcag acacaagaac attgtacgct tcttgggatgttgttggaac 258O aagaac acta gacittctitat gtatgattat atgtcaaatg ggagtttggg aagtttgctt 264 O cacgaaagga gcggtgitatg tag cittagga tigggaagtga ggtacaagat tatacttggit 27 OO gcagotcagg gtttggctta cittgcaccat gactgttgttc citcc.cattgttcatagagac 276 O. atcaaggcaa ataatattot gattggcc ct gattittgaac cittacattgg agattitcgga 282O citc.gctaagc titgttgatga tiggcg actitt gctic gttctt coaacac cat tdctggttcc 2880 tatggittaca tagctocagg tactitcccaa atttctgttt tactgctaat tagittaactt 2.940 gata catcat aagttactag tagtottagc ctagtaatgg tagaaatto a ggitttatagt 3OOO ttggittittgg tttittgg cat tittgaaaaga atagtattitt g gttcagttg attittcg att 3060 tggtttaaaa ttittagaaaa aaaatggtag aagaaataat ttittgattaa tattatato a 312 O totact.gitat acg atttaaa aaaaaaattg atttggitttg gtctogatat tittgcttgta 318O gcttggttca atatttittac tatatatatg attcatgact titcatactaatcaatatttg 3240 titcggitttcg ttittgattag totgaaagtg aatcatatag ttaaaatatt gaccaatcta 33OO toatatactt acacgctaca agattittcga tigtaattittg gtgtaag cat tittggitttitt 3360 atatgttcat cottagtaac taacaacaaa tatcaattgg gaag cataac gittatatatt 342O cc cataaggc tatalacacta taattatgca gaatacggat act caatgaa gataacagag 3480 aaaag.cgacg totacagota toggagtcgta gtgctic gagg tactaacggg taagcaacca 354. O atcgatccaa cqataccaga tigg actocac atagtgg act ggg to aagaa aatcagagac 3600 atacaagtaa to gaccalagg act acaag.ca agaccagagt cagaggttga agagatgatg 3660 caaacgctag gag togcgct tittatgcatt aatccaatac cc galagacag goctacaatg 372 O aaagatgtgg citgctatoct tagtgagata totcaa.gaga gagaggaatc gatgaaagtt 378 O. gatggttgct c gggaagttg taacaatgga agagaacgtg gcaaagatga titcgactitca 384 O toggittatgc aacaaacggc taagtatttg agaagtagta gcacgagttt citctg.cgtot 39 OO totttgctitt acticttctitc ttcttctgct acttctaatg ttagaccaaa tottaaataa 396 O
<210> SEQ ID NO 4 &2 11s LENGTH 1135 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 4 Met Ser Leu Gln Met Pro Ile Pro Arg Lys Lys Ala Leu Thr Val Ser 1 5 10 15
His Phe Ser Ile Thr Leu Ser Leu Phe Leu Ala Phe Phe Ile Ser Ser 2O 25 30 Thr Ser Ala Ser Thr Asn Glu Val Ser Ala Leu Ile Ser Trp Leu. His 35 40 45 US 2004/OO67506 A1 Apr. 8, 2004 45
-continued
Ser Ser Asn Ser Pro Pro Pro Ser Val Phe Ser Gly Trp Asin Pro Ser 50 55 60 Asp Ser Asp Pro Cys Gln Trp Pro Tyr Ile Thr Cys Ser Ser Pro Asp 65 70 75 8O Asn Lys Lieu Val Thr Glu Ile Asn Val Val Ser Val Glin Leu Ala Lieu 85 90 95 Pro Phe Pro Pro Asn Ile Ser Ser Phe Thr Ser Leu Gln Lys Leu Val 100 105 110 Ile Ser Asn. Thir Asn Lieu. Thr Gly Ala Ile Ser Ser Glu Ile Gly Asp 115 120 125 Cys Ser Glu Lieu. Ile Val Ile Asp Leu Ser Ser Asn. Ser Lieu Val Gly 130 135 1 4 0 Glu Ile Pro Ser Ser Lieu Gly Lys Lieu Lys Asn Lieu Glin Glu Lieu. Cys 145 15 O 155 160 Lieu. Asn. Ser Asn Gly Lieu. Thr Gly Lys Ile Pro Pro Glu Lieu Gly Asp 1.65 170 175 Cys Val Ser Lieu Lys Asn Lieu Glu Ile Phe Asp Asn Tyr Lieu Ser Glu 18O 185 190 Asn Lieu Pro Leu Glu Lieu Gly Lys Ile Ser Thr Lieu Glu Ser Ile Arg 195 200 2O5 Ala Gly Gly Asn. Ser Glu Lieu Ser Gly Lys Ile Pro Glu Glu Ile Gly 210 215 220 Asn. Cys Arg Asn Lieu Lys Wall Leu Gly Lieu Ala Ala Thir Lys Ile Ser 225 230 235 240 Gly Ser Lieu Pro Val Ser Leu Gly Glin Leu Ser Lys Lieu Glin Ser Lieu 245 250 255 Phe Val Tyr Ser Thr Met Leu Ser Gly Glu Ile Pro Lys Glu Leu Gly 260 265 27 O Asn. Cys Ser Glu Lieu. Ile Asn Lieu Phe Leu Tyr Asp Asn Asp Leu Ser 275 280 285 Gly Thr Lieu Pro Lys Glu Lieu Gly Lys Lieu Glin Asn Lieu Glu Lys Met 29 O 295 3OO Leu Lleu Trp Glin Asn. Asn Lieu. His Gly Pro Ile Pro Glu Glu Ile Gly 305 310 315 320 Phe Met Lys Ser Leu Asn Ala Ile Asp Leu Ser Met Asn Tyr Phe Ser 325 330 335 Gly. Thir Ile Pro Lys Ser Phe Gly Asn Lieu Ser Asn Lieu Glin Glu Lieu 340 345 350 Met Leu Ser Ser Asn Asn Ile Thr Gly Ser Ile Pro Ser Ile Leu Ser 355 360 365 Asp Cys Thr Lys Lieu Val Glin Phe Glin Ile Asp Ala Asn Glin Ile Ser 370 375 38O Gly Lieu. Ile Pro Pro Glu Ile Gly Lieu Lleu Lys Glu Lieu. Asn. Ile Phe 385 390 395 400 Leu Gly Trp Glin Asn Lys Lieu Glu Gly Asn. Ile Pro Asp Glu Lieu Ala 405 410 415 Gly Cys Glin Asn Lieu Glin Ala Lieu. Asp Leu Ser Glin Asn Tyr Lieu. Thr 420 425 430 Gly Ser Lieu Pro Ala Gly Lieu Phe Glin Leu Arg Asn Lieu. Thir Lys Lieu 435 4 40 4 45 US 2004/OO67506 A1 Apr. 8, 2004 46
-continued Leu Lieu. Ile Ser Asn Ala Ile Ser Gly Val Ile Pro Leu Glu Thr Gly 450 455 460 Asn. Cys Thir Ser Lieu Val Arg Lieu Arg Lieu Val Asn. Asn Arg Ile Thr 465 470 475 480 Gly Glu Ile Pro Lys Gly Ile Gly Phe Leu Glin Asn Lieu Ser Phe Lieu 485 490 495 Asp Leu Ser Glu Asn. Asn Lieu Ser Gly Pro Val Pro Leu Glu Ile Ser 5 OO 505 510 Asn. Cys Arg Glin Leu Gln Met Lieu. Asn Lieu Ser Asn. Asn. Thir Lieu Glin 515 52O 525 Gly Tyr Lieu Pro Leu Ser Leu Ser Ser Lieu. Thir Lys Lieu Glin Val Lieu 530 535 540 Asp Wal Ser Ser Asn Asp Lieu. Thr Gly Lys Ile Pro Asp Ser Lieu Gly 545 550 555 560 His Lieu. Ile Ser Lieu. Asn Arg Lieu. Ile Leu Ser Lys Asn. Ser Phe Asn 565 570 575 Gly Glu Ile Pro Ser Ser Leu Gly His Cys Thr Asn Lieu Glin Leu Lieu 58O 585 59 O Asp Leu Ser Ser Asn. Asn. Ile Ser Gly Thir Ile Pro Glu Glu Lieu Phe 595 600 605 Asp Ile Glin Asp Leu Asp Ile Ala Lieu. Asn Lieu Ser Trp Asn. Ser Lieu 610 615 62O Asp Gly Phe Ile Pro Glu Arg Ile Ser Ala Lieu. Asn Arg Lieu Ser Val 625 630 635 640 Leu Asp Ile Ser His Asn Met Leu Ser Gly Asp Leu Ser Ala Leu Ser 645 650 655 Gly Lieu Glu Asn Lieu Val Ser Lieu. Asn. Ile Ser His Asn Arg Phe Ser 660 665 670 Gly Tyr Lieu Pro Asp Ser Lys Val Phe Arg Glin Lieu. Ile Gly Ala Glu 675 680 685 Met Glu Gly Asn. Asn Gly Lieu. Cys Ser Lys Gly Phe Arg Ser Cys Phe 69 O. 695 7 OO Val Ser Asn Ser Ser Gln Leu Thir Thr Glin Arg Gly Val His Ser His 705 710 715 720 Arg Lieu Arg Ile Ala Ile Gly Lieu Lieu. Ile Ser Val Thr Ala Val Lieu 725 730 735 Ala Wall Leu Gly Val Lieu Ala Val Ile Arg Ala Lys Gln Met Ile Arg 740 745 750 Asp Asp Asn Asp Ser Glu Thr Gly Glu Asn Lieu Trp Thir Trp Glin Phe 755 760 765 Thr Pro Phe Gln Lys Leu Asn Phe Thr Val Glu His Val Leu Lys Cys 770 775 78O Leu Val Glu Gly Asn Val Ile Gly Lys Gly Cys Ser Gly Ile Val Tyr 785 790 795 8OO Lys Ala Glu Met Pro Asn Arg Glu Val Ile Ala Wall Lys Lys Lieu Trp 805 810 815 Pro Val Thr Val Pro Asn Leu Asn Glu Lys Thr Lys Ser Ser Gly Val 820 825 830 Arg Asp Ser Phe Ser Ala Glu Wall Lys Thr Lieu Gly Ser Tle Arg His 835 840 845 Lys Asn. Ile Val Arg Phe Leu Gly Cys Cys Trp Asn Lys Asn. Thir Arg US 2004/OO67506 A1 Apr. 8, 2004 47
-continued
85 O 855 860 Leu Lleu Met Tyr Asp Tyr Met Ser Asn Gly Ser Leu Gly Ser Lieu Lieu 865 870 875 88O His Glu Arg Ser Gly Val Cys Ser Lieu Gly Trp Glu Val Arg Tyr Lys 885 890 895 Ile Ile Leu Gly Ala Ala Glin Gly Lieu Ala Tyr Lieu. His His Asp Cys 9 OO 905 910 Val Pro Pro Ile Val His Arg Asp Ile Lys Ala Asn. Asn. Ile Lieu. Ile 915 920 925 Gly Pro Asp Phe Glu Pro Tyr Ile Gly Asp Phe Gly Lieu Ala Lys Lieu 930 935 940 Val Asp Asp Gly Asp Phe Ala Arg Ser Ser Asn. Thir Ile Ala Gly Ser 945 950 955 96.O Tyr Gly Tyr Ile Ala Pro Glu Tyr Gly Tyr Ser Met Lys Ile Thr Glu 965 970 975 Lys Ser Asp Val Tyr Ser Tyr Gly Val Val Val Leu Glu Val Leu Thr 98O 985 99 O Gly Lys Glin Pro Ile Asp Pro Thir Ile Pro Asp Gly Lieu. His Ile Val 995 10 OO 1005
Asp W a. Lys Lys Ile Arg Asp Ile Glin Val Ile Asp Glin Gly O 10 O15 O20 Leu Glin Ala Arg Pro Glu Ser Glu Val Glu Glu Met Met Glin Thr O25 O3O O35 Leu Gly Val Ala Lieu Lieu. Cys Ile Asn Pro Ile Pro Glu Asp Arg
Pro h Met Lys Asp Val Ala Ala Met Leu Ser Glu Ile Cys Glin
Glu Arg Glu Glu Ser Met Lys Val Asp Gly Cys Ser Gly Ser Cys
Asn Asn Gly Arg Glu Arg Gly Lys Asp Asp Ser Thr Ser Ser Val
Met Glin Gln Thr Ala Lys Tyr Leu Arg Ser Ser Ser Thr Ser Phe
Ser Ala Ser Ser Leu Leu Tyr Ser Ser Ser Ser Ser Ala Thr Ser 115 120 125 Asn Val Arg Pro Asn Lieu Lys 130 135
<210 SEQ ID NO 5 &2 11s LENGTH 25 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 5 Arg Tyr Lys Ile Ile Leu Gly Ala Ala Glin Gly Lieu Ala Tyr Lieu. His 1 5 10 15 His Asp Cys Val Pro Pro Ile Val His 2O 25
<210> SEQ ID NO 6 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana &220s FEATURE
US 2004/OO67506 A1 Apr. 8, 2004 49
-continued Carythamcg gwgaaathcc 20
3. 2 1 O > S EQ ID NO 8 ENGTH 2.0 YPE DNA RGANISM: Arabidopsis thaliana EATURE NAM E/KEY: misc feature O ATION: (1) . . (1) ER INFORMATION m = a or c
misc feature (3) . . (3) ORMATION: y = c or t misc feature (4) . . (4) ORMATION: y = c or t
3. 2 2 NAM E A K E Y misc feature O CA. T I O N (6) . . (6) ORMATION: s = g or c misc feature (8) ... (8) ORMATION: v = a or g or c misc feature (9) ... (9) ORMATION: y = c or t misc feature (13) . . (13) ORMATION: h = a or C or it
misc feature 3. 2 2 2 > O CA. T I O N (18) . . (18) ORMATION: h = a or c or it
<400 SEQUENCE: 8 mayytsavyg gahctathcc 20
EQ ID NO 9 ENGTH 22 YPE DNA RGANISM: Arabidopsis thaliana EATURE <221 E/KEY: misc feature <222> LOCATION: (2) ... (2) &223> OTHER INFORMATION: h = a or c or it &220s FEATURE <221 AKEY misc feature <222> L C T I O N (5) . . (5) &223> OTH ORMATION: W = a or it &220s F <221 misc feature <222> L C T I O N (8) ... (8) &223> OTH ORMATION: W = a or it &220s F <221 misc feature <222> L C T I O N (10) . . (10) &223> OTH ORMATION: s = g or c &220s FEATURE <221 NAME/KEY misc feature <222> LOCATION (17) . . (17) &223> OTHER IN ORMATION: n = a, c, g or t <400 SEQUENCE: 9 thccwttwigs catgtantica ta 22
<210> SEQ ID NO 10 US 2004/OO67506 A1 Apr. 8, 2004 50
-continued
&2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: y = c or t &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (5) . . (5) <223> OTHER INFORMATION: d = a or g or t &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (8) ... (8) <223> OTHER INFORMATION: d = a or g or t &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (11) . . (11) &223> OTHER INFORMATION: h = a or c or it &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (13) . . (13) &223> OTHER INFORMATION: h = a or c or it &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (17) . . (17) <223> OTHER INFORMATION: d = a or g or t <400 SEQUENCE: 10 aytcdggdgc hahgtadcc 19
<210> SEQ ID NO 11 &211's LENGTH 21 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (1) . . (1) &223> OTHER INFORMATION m = a or c &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (3) . . (3) <223> OTHER INFORMATION: y = c or t &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (4) ... (4) &223> OTHER INFORMATION: h = a or c or it &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (7) . . (7) <223> OTHER INFORMATION: r = g or a &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (10) . . (10) <223> OTHER INFORMATION: r = g or a &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (13) . . (13) <223> OTHER INFORMATION: r = g or a &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (16) . . (16) <223> OTHER INFORMATION: v = a or g or c <400 SEQUENCE: 11 mayhccraar ctrtavacat c 21
<210> SEQ ID NO 12 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana US 2004/OO67506 A1 Apr. 8, 2004 51
-continued <400 SEQUENCE: 12 cgatcagaca caagaacat 19
<210> SEQ ID NO 13 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 13 agcaatggtg ttggaagaa 19
<210> SEQ ID NO 14 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (11) . . (11) <223> OTHER INFORMATION: y = c or t <400 SEQUENCE: 14 tgcagatctt ydtgaagac 19
<210 SEQ ID NO 15 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400s. SEQUENCE: 15 gacticcittct g gatgttg 18
<210> SEQ ID NO 16 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 16 tgtaaaacga cqgccagt 18
<210 SEQ ID NO 17 <211& LENGTH: 34 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 17 cacacaggat coaatcggca tttgcaaaga cata 34
<210> SEQ ID NO 18 <211& LENGTH: 842 &212> TYPE DNA <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 18 aagcttaaat gacaatttag taccttgggit togtoatgat ttagagcgga acaaatatac 60 catacatcaa acgaggatat acagagaaaa ttcatggaag tatggaattt agaggacaat 120 ttct cittctg. g.gctacaacg gaccggcc.ca titcgctcatt tacccagagg tatcgagttt 18O gtgg acttitt gatgcc.gcta gagacitattg gcatcggatt gaaaaaaatg titt actt.cgt. 240 tgttaacaat tittctgaatg caatatttitc cittgtcatga atatttaaac ttgttattac 3OO US 2004/OO67506 A1 Apr. 8, 2004 52
-continued tittcttittag cittaggtgtg gacaattatg gagtttacitt caaacgagga agaatcttaa 360 acgctoggtt caggtotcga aaacaaacca acticacaatc citgacittaat tdaggaaaac 420 aatgcaaaac cacatgcatg ctitccatatt totatoataa tottataaga aaaaacacta 480 ctaagttgaaa tdattctgta tatatataac caatgccttt tattttgttga tattittatgt 540 atatataact attgacttitt gtdatctatg gatagtgtct cqggctott g g caaacatat 600 ttcaaagaaa agittaatgac totaattaat taatctgaag citagaaacag aaccocq agg 660 taaaagaaaa agacagagca catgaagttt agtacttitta tatatttaat atato attct 720 ttcttattgc titatctotaa agcaaaaact tcc ctaaacc ctaagccaaa g g acticagat 78O cgatgcagaa ccaagaaggc titgttittgga tittgaga.gcc aaatgcaaag aaaaaaactic 840 tt 842
<210 SEQ ID NO 19 &2 11s LENGTH 1130 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 19 Met Pro Ile Pro Arg Lys Lys Ala Leu Thr Val Ser His Phe Ser Ile 1 5 10 15
Thir Leu Ser Leu Phe Leu Ala Phe Phe Ile Ser Ser Thr Ser Ala Ser 2O 25 30 Thr Asn Glu Val Ser Ala Leu Ile Ser Trp Leu His Ser Ser Asn Ser 35 40 45 Pro Pro Pro Ser Val Phe Ser Gly Trp Asin Pro Ser Asp Ser Asp Pro 50 55 60 Cys Glin Trp Pro Tyr Ile Thr Cys Ser Ser Pro Asp Asn Lys Leu Val 65 70 75 8O
Thr Glu Ile Asn. Wal Wal Ser Wall Glin Leu Ala Leu Pro Phe Pro Pro 85 90 95 Asn Ile Ser Ser Phe Thr Ser Leu Gln Lys Leu Val Ile Ser Asn Thr 100 105 110 Asn Lieu. Thr Gly Ala Ile Ser Ser Glu Ile Gly Asp Cys Ser Glu Lieu 115 120 125 Ile Val Ile Asp Leu Ser Ser Asn Ser Leu Val Gly Glu Ile Pro Ser 130 135 1 4 0 Ser Lieu Gly Lys Lieu Lys Asn Lieu Glin Glu Lieu. Cys Lieu. Asn. Ser Asn 145 15 O 155 160 Gly Lieu. Thr Gly Lys Ile Pro Pro Glu Lieu Gly Asp Cys Wal Ser Lieu 1.65 170 175 Lys Asn Lieu Glu Ile Phe Asp Asn Tyr Lieu Ser Glu Asn Lieu Pro Leu 18O 185 190 Glu Lieu Gly Lys Ile Ser Thr Lieu Glu Ser Ile Arg Ala Gly Gly Asn 195 200 2O5 Ser Glu Lieu Ser Gly Lys Ile Pro Glu Glu Ile Gly Asn. Cys Arg Asn 210 215 220 Leu Lys Val Lieu Gly Lieu Ala Ala Thr Lys Ile Ser Gly Ser Lieu Pro 225 230 235 240 Val Ser Leu Gly Gln Leu Ser Lys Leu Glin Ser Leu Phe Val Tyr Ser 245 250 255 US 2004/OO67506 A1 Apr. 8, 2004 53
-continued Thr Met Leu Ser Gly Glu Ile Pro Lys Glu Lieu Gly Asn. Cys Ser Glu 260 265 27 O Lieu. Ile Asn Lieu Phe Leu Tyr Asp Asn Asp Leu Ser Gly Thr Lieu Pro 275 280 285 Lys Glu Lieu Gly Lys Lieu Glin Asn Lieu Glu Lys Met Lieu Lleu Trp Glin 29 O 295 3OO Asn Asn Leu. His Gly Pro Ile Pro Glu Glu Ile Gly Phe Met Lys Ser 305 310 315 320 Leu Asn Ala Ile Asp Leu Ser Met Asn Tyr Phe Ser Gly Thr Ile Pro 325 330 335 Lys Ser Phe Gly Asn Lieu Ser Asn Lieu Glin Glu Lieu Met Leu Ser Ser 340 345 350 Asn Asn. Ile Thr Gly Ser Ile Pro Ser Ile Leu Ser Asp Cys Thr Lys 355 360 365 Leu Val Glin Phe Glin Ile Asp Ala Asn Glin Ile Ser Gly Lieu. Ile Pro 370 375 38O Pro Glu Ile Gly Lieu Lleu Lys Glu Lieu. Asn. Ile Phe Gly Trp Glin Asn 385 390 395 400 Lys Lieu Glu Gly Asn. Ile Pro Asp Glu Lieu Ala Gly Cys Glin Asn Lieu 405 410 415 Glin Ala Lieu. Asp Leu Ser Glin Asn Tyr Lieu. Thr Gly Ser Lieu Pro Ala 420 425 430 Gly Lieu Phe Glin Leu Arg Asn Lieu. Thir Lys Lieu Lleu Lieu. Ile Ser Asn 435 440 445 Ala Ile Ser Gly Val Ile Pro Leu Glu Thr Gly Asn Cys Thr Ser Leu 450 455 460 Val Arg Lieu Arg Lieu Val Asn. Asn Arg Ile Thr Gly Glu Ile Pro Lys 465 470 475 480 Gly Ile Gly Phe Leu Glin Asn Lieu Ser Phe Lieu. Asp Lieu Ser Glu Asn 485 490 495 Asn Lieu Ser Gly Pro Val Pro Leu Glu Ile Ser Asn. Cys Arg Glin Lieu 5 OO 505 510 Glin Met Lieu. Asn Lieu Ser Asn. Asn. Thir Lieu Glin Gly Tyr Lieu Pro Leu 515 52O 525 Ser Lieu Ser Ser Lieu. Thir Lys Lieu Glin Val Lieu. Asp Wal Ser Ser Asn 530 535 540 Asp Lieu. Thr Gly Lys Ile Pro Asp Ser Lieu Gly His Lieu. Ile Ser Lieu 545 550 555 560 Asn Arg Lieu. Ile Leu Ser Lys Asn. Ser Phe Asn Gly Glu Ile Pro Ser 565 570 575 Ser Lieu Gly. His Cys Thr Asn Lieu Glin Lieu Lieu. Asp Leu Ser Ser Asn 58O 585 59 O Asn. Ile Ser Gly Thr Ile Pro Glu Glu Lieu Phe Asp Ile Glin Asp Lieu 595 600 605 Asp Ile Ala Lieu. Asn Lieu Ser Trp Asn. Ser Lieu. Asp Gly Phe Ile Pro 610 615 62O Glu Arg Ile Ser Ala Lieu. Asn Arg Lieu Ser Val Lieu. Asp Ile Ser His 625 630 635 640 Asn Met Leu Ser Gly Asp Leu Ser Ala Leu Ser Gly Lieu Glu Asn Lieu 645 650 655 Val Ser Lieu. Asn. Ile Ser His Asn Arg Phe Ser Gly Tyr Lieu Pro Asp US 2004/OO67506 A1 Apr. 8, 2004 54
-continued
660 665 670 Ser Lys Val Phe Arg Glin Lieu. Ile Gly Ala Glu Met Glu Gly Asn. Asn 675 680 685 Gly Lieu. Cys Ser Lys Gly Phe Arg Ser Cys Phe Val Ser Asn. Ser Ser 69 O. 695 7 OO Glin Lieu. Thir Thr Glin Arg Gly Val His Ser His Arg Lieu Arg Ile Ala 705 710 715 720 Ile Gly Leu Leu Ile Ser Val Thr Ala Val Leu Ala Val Leu Gly Val 725 730 735 Leu Ala Val Ile Arg Ala Lys Glin Met Ile Arg Asp Asp Asn Asp Ser 740 745 750 Glu Thr Gly Glu Asn Leu Trp Thr Trp Glin Phe Thr Pro Phe Glin Lys 755 760 765 Lieu. Asn. Phe Thr Val Glu His Val Lieu Lys Cys Lieu Val Glu Gly Asn 770 775 78O Val Ile Gly Lys Gly Cys Ser Gly Ile Val Tyr Lys Ala Glu Met Pro 785 790 795 8OO Asn Arg Glu Val Ile Ala Val Lys Lys Leu Trp Pro Val Thr Val Pro 805 810 815 Asn Lieu. Asn. Glu Lys Thr Lys Ser Ser Gly Val Arg Asp Ser Phe Ser 820 825 830 Ala Glu Wall Lys Thr Lieu Gly Ser Ile Arg His Lys Asn. Ile Val Arg 835 840 845 Phe Leu Gly Cys Cys Trp Asn Lys Asn. Thir Arg Lieu Lleu Met Tyr Asp 85 O 855 860 Tyr Met Ser Asn Gly Ser Leu Gly Ser Lieu Lieu. His Glu Arg Ser Gly 865 870 875 88O Val Cys Ser Lieu Gly Trp Glu Val Arg Tyr Lys Ile Ile Leu Gly Ala 885 890 895 Ala Glin Gly Lieu Ala Tyr Lieu. His His Asp Cys Val Pro Pro Ile Val 9 OO 905 910 His Arg Asp Ile Lys Ala Asn. Asn. Ile Lieu. Ile Gly Pro Asp Phe Glu 915 920 925 Pro Tyr Ile Gly Asp Phe Gly Lieu Ala Lys Lieu Val Asp Asp Gly Asp 930 935 940 Phe Ala Arg Ser Ser Asn. Thir Ile Ala Gly Ser Tyr Gly Tyr Ile Ala 945 950 955 96.O Pro Glu Tyr Gly Tyr Ser Met Lys Ile Thr Glu Lys Ser Asp Val Tyr 965 970 975 Ser Tyr Gly Val Val Val Leu Glu Val Leu Thr Gly Lys Glin Pro Ile 98O 985 99 O Asp Pro Thir Ile Pro Asp Gly Lieu. His Ile Val Asp Trp Val Lys Lys 995 10 OO 1005 Ile Arg Asp Ile Glin Val Ile Asp Glin Gly Lieu Glin Ala Arg Pro 1010 1015 1020 Glu Ser Glu Val Glu Glu Met Met Gln Thr Leu Gly Val Ala Leu 1025 1030 1035 Lieu. Cys Ile Asn Pro Ile Pro Glu Asp Arg Pro Thr Met Lys Asp 1040 1045 105 O Val Ala Ala Met Leu Ser Glu Ile Cys Glin Glu Arg Glu Glu Ser 1055 1060 1065 US 2004/OO67506 A1 Apr. 8, 2004
-continued
Met Lys Val Asp Gly Cys Ser Gly Ser Cys Asn. Asn Gly Arg Glu 1070 1075 1080
Arg Gly Lys Asp Asp Ser Thr Ser Ser Wal Met Glin Glin Thr Ala 1085 1090 1095
Lys Tyr Leu Arg Ser Ser Ser Thr Ser Phe Ser Ala Ser Ser Leu 1100 1105 1110 Leu Tyr Ser Ser Ser Ser Ser Ala Thr Ser Asn Val Arg Pro Asn 1115 1120 1125 Leu Lys 1130
1. An isolated nucleic acid comprising a transcriptional provided that said nucleic acid is not one of the nucleic regulatory root promoter or a functional fragment of Such a acids as deposited under the GenBank Accession num promoter and/or comprising a nucleic acid Sequence encod bers ABO 17061, AV538507, AV520681 or AQ966419. ing a novel LRR receptor-like kinase (protein) or an immu 2. An isolated nucleic acid comprising a transcriptional nologically active and/or functional fragment of Such a regulatory root promoter comprising the Sequence as given protein (kinase) Selected from the group consisting of: in any of SEQ ID NOs 1, 2 or 18. (a) a nucleic acid comprising the DNA sequence as given 3. An isolated nucleic acid comprising a transcriptional in any of SEQID NOS 1, 2, 3 or 18, or the complement regulatory root promoter consisting of the Sequence as given thereof, in SEQID NO 2 or 18, or a functional part of said sequence which is able to regulate gene expression in a root Specific (b) a nucleic acid comprising the RNA sequences corre manner, provided that Said nucleic acid is not one of the sponding to any of SEQ ID NOS 1, 2, 3 or 18, or the nucleic acids as deposited under the GenBank Accession complement thereof, numbers AV538507 or AV52O681. (c) a nucleic acid specifically hybridizing with the nucle 4. An isolated nucleic acid comprising a transcriptional otide sequence as defined in (a) or (b), regulatory root Specific promoter comprising: (d) a nucleic acid encoding a protein comprising the (a) at least a functional part of the DNA sequence as given amino acid sequence as given in SEQ ID NO 4, SEQ in any of SEQ ID NO 1, 2 or 18, and, ID NO 5 or SEQ ID NO 19, (b) a Second transcriptional regulatory Sequence, option (e) a nucleic acid which is degenerated, as a result of the ally a regulatory promoter Sequence not normally genetic code, to a nucleotide Sequence as defined in any exhibiting root-Specificity. of (a) to (d), and which Sequence codes for a protein 5. An isolated nucleic acid according to claim 4 wherein having the amino acid sequence as given in SEQID NO Said Second transcriptional regulatory promoter Sequence is 4, SEQ ID NO 19, the ubiquitin promoter. (f) a nucleic acid which is diverging, due to the differences 6. An isolated nucleic acid Selected from the group in codon usage between the organisms, to a nucleotide consisting of: Sequence as defined in any of (a) to (d), and which (a) a nucleic acid consisting of at least part of the DNA Sequence codes for a protein having the amino acid sequence as given in SEQ ID NO 1 or 3, or the sequence as given in SEQ ID NO 4, SEQ ID NO 19, complement thereof, (g) a nucleic acid which is diverging, due to the differ ences between alleles, to a nucleotide Sequence as (b) a nucleic acid encoding a protein as given in SEQ ID defined in any of (a) to (d), and which sequence codes NO 4 or 19 or encoding a fragment of Said protein, for a protein having the amino acid Sequence as given wherein Said fragment comprises the Sequence as rep in SEQ ID NO 4, SEQ ID NO 19, resented in SEQ ID NO 5, and (h) a nucleic acid encoding a fragment of a protein (c) a nucleic acid encoding a protein with an amino acid encoded by a DNA sequence as given in SEQ ID NO Sequence which is at least 65% identical to the protein 1 or SEQ ID NO3 or encoding a fragment of a protein as given in SEQ ID NO 4 or 9, wherein said amino acid encoded by a nucleic acid as defined in any one of (a) Sequence comprises the Sequence as represented in to (g), wherein said fragment comprises the sequence SEO ID NO 5, characterised in that said nucleic acid as represented in SEQ ID NO 5, and, encodes a novel LRR receptor-like kinase protein or an immunologically active and/or functional fragment of (k) a nucleic acid encoding a protein as defined in SEQID Such a protein, and further provided that Said nucleic NO 4, SEQ ID NO 19 or a nucleic acid as defined in acid is not one of the nucleic acids as deposited under any one of (a) to (h), Said nucleic acid interrupted by the GenBank Accession numbers ABO17061 or intervening DNA, AO966419. US 2004/OO67506 A1 Apr. 8, 2004 56
7. An isolated nucleic acid according to any of claims 1 22. A transgenic plant cell comprising a nucleic acid to 6 which is DNA, cDNA, genomic DNA, synthetic DNA, Sequence of any of claims 1 to 7 which is operably linked to or RNA wherein T is replaced by U. regulatory elements allowing transcription and/or expres 8. A nucleic acid molecule of at least 15 nucleotides in Sion of Said nucleic acid in plant cells or obtainable by a length hybridizing Specifically with a nucleic acid of any of method of claim 20 or 21. claims 2 to 7. 23. The transgenic plant cell of claim 22 wherein said 9. A nucleic acid molecule of at least 15 nucleotides in nucleic acid of any of claims 1 to 7 is stably integrated into length Specifically amplifying a nucleic acid of any of claims the genome of Said plant cell. 2 to 7. 24. A transgenic plant or plant tissue comprising plant 10. A vector comprising a nucleic acid Sequence accord cells of claim 22 or 23. ing to any of claims 1 to 7. 25. A harvestable part of a plant of claim 24. 11. A vector according to claim 10 which is an expression 26. The harvestable part of claim 25 which is selected vector wherein Said nucleic acid Sequence encoding a novel from the group consisting of Seeds, leaves, fruits, Stem LRR receptor-like kinase or an immunologically active cultures, rhizomes, tubers and bulbs. and/or functional fragment thereof, is operably linked to one 27. The progeny derived from any of the plants or plant or more control Sequences allowing the expression in parts of any of claims 24 to 26. prokaryotic and/or eukaryotic host cells. 28. A method for conferring root-specificity, root-mer 12. A vector according to claim 10 wherein Said vector istem-specificity, root-Vascular-tissue-specificity, root-endo comprises at least part of a nucleic acid according to any of dermis-specificity, root-cortex-Specificity, root-epidermis claims 2 to 7 and wherein Said transcriptional regulatory root Specificity, root-quiescent-center-specificity, and/or promoter or functional parts thereof is operably linked to abundant expression in root and/or early expression in root one or more genes of interest. to other promoter Sequences comprising the fusion at least a 13. A host cell containing a nucleic acid molecule accord functional part of the DNA sequence as given in SEQID NO ing to any of claim 1 to 7 or a vector according to any of 1, 2 or 18 to a Second transcriptional regulatory promoter claims 10 to 12. Sequence normally not exhibiting root-specificity. 14. The host cell according to claim 13, wherein said host 29. A method for root-specific expression of a gene(s) of cell is a bacterial, insect, fungal, plant or animal cell. interest comprising operably linking of Said gene(s) of 15. An isolated LRR receptor-like kinase comprising one interest to a transcriptional regulatory root-specific promoter of the polypeptides Selected from the group consisting of as defined in any of claims 2 to 5. 30. A method for modifying cell fate and/or plant devel (a) a polypeptide as given in SEQID NO 4 or SEQ ID NO opment and/or plant morphology and/or plant biochemistry 19, and/or plant physiology comprising the modification of (b) a polypeptide comprising the amino acid sequence as expression in the meristem of the main and the lateral roots, given in SEQ ID NO 5, and, in the vascular tissue of the root or in the lateral root primordia of a gene(s) of interest operably linked to a (c) a polypeptide encoded by a nucleic acid as given in transcriptional regulatory root-Specific promoter as defined SEQ ID NO 1 or 3, in any of claims 2 to 5. or a homologue or a derivative of Said protein, or an 31. A method for phytoremediation comprising the immunologically active and/or functional fragment expression of a gene(s) of Interest under the control of a thereof. transcriptional regulatory root-Specific promoter as defined 16. A polypeptide as defined in claim 15 being a root in any of claims 2 to 5. CLAVATA homologue or a functional homologue thereof 32. A method for environmental remediation comprising 17. An isolated polypeptide comprising the Sequence the expression of a gene(s) of interest under the control of a represented in SEQ ID NO 5 encodable by a nucleic acid of transcriptional regulatory root-Specific promoter as defined any of claims 1 to 7, or a homologue or a derivative thereof, in any of claims 2 to 5. or an immunologically active and/or functional fragment 33. A method for enhancing plant growth and crop yield thereof. comprising the expression of a gene(s) of interest under the 18. A method for producing a polypeptide according to control of a a transcriptional regulatory root-Specific pro any of claims 15 to 17 comprising culturing a host cell of moter as defined in any of claims 2 to 5. claim 13 or 14 under conditions allowing the expression of 34. A method for conferring enhanced resistance to patho the polypeptide and recovering the produced polypeptide gens which attack the belowground plant tissue comprising from the culture. the expression of a gene(s) of interest under the control of a 19. An antibody Specifically recognizing an LRR recep transcriptional regulatory root-Specific promoter as defined tor-like kinase (protein) of any of claims 15 to 17 or in any of claims 2 to 5. recognizing immunologically active parts or Specific 35. A method for enhancing freezing tolerance in plants epitopes thereof. comprising the expression of a gene(s) of interest under the 20. A method for the production of transgenic plants, plant control of a transcriptional regulatory root-Specific promoter cells or plant tissues comprising the introduction of a nucleic as defined in any of claims 2 to 5. acid molecule according to any of claims 1 to 7 in an 36. A method for enhancing resistance of a plant to expressible format or a vector according to any of claims 10 drought and/or high Salt comprising the expression of a to 12 in Said plant, plant cell or plant tissue. gene(s) of interest under the control of a transcriptional 21. The method of claim 20 further comprising regener regulatory root-specific promoter as defined in any of claims ating a plant from Said plant cell. 2 to 5. US 2004/OO67506 A1 Apr. 8, 2004 57
37. A method for the production of light-sensitive proteins or organs of a plant, of a nucleic acid encoding an LRR comprising the expression of a gene encoding Said light receptor-like kinase operably linked to a plant-operable Sensitive protein under the control of a transcriptional regu promoter Sequence. latory root-specific promoter as defined in any of claims 2 to 50. A method for enhancing root formation and/or root 5. growth comprising the expression in particular cells, tissues 38. A method for root Specific gene Silencing comprising or organs of a plant, of a nucleic acid encoding an LRR the expression of an antisense nucleotide Sequence to the receptor-like kinase as defined in any of claims 15 to 17 gene of interest, a gene Silencing construct or a ribozyme under the control of a plant operable promoter Sequence. under the control of a transcriptional regulatory root-Specific 51. A method for enhancing root formation and/or root promoter as defined in any of claims 2 to 5. growth comprising the expression in particular cells, tissues or organs of a plant, of a nucleic acid encoding an LRR 39. A method for stimulating root meristem formation receptor-like kinase under the control of a transcriptional comprising the expression of a gene that influences the regulatory root promoter as defined in any of claims 2 to 5. proliferation and/or differentiation of the root meristem 52. A method for enhancing root-formation and/or root under the control of a transcriptional regulatory root-Specific growth comprising the expression in particular cells, tissues promoter as defined in any of claims 2 to 5. or organs of a plant, of a nucleic acid encoding a polypeptide 40. A method for stimulating root meristem formation as defined in any of claims 15 to 17 under the control of a comprising the expression in particular cells, tissueS or transcriptional regulatory root promoter as defined in any of organs of a plant, of a nucleic acid encoding an LRR claims 2 to 5. receptor-like kinase operably linked to a plant-operable 53. A method for enhancing overall growth and yield promoter Sequence. comprising the expression in particular cells, tissueS or 41. A method Stimulating root meristem formation com organs of a plant, of a nucleic acid encoding an LRR prising the expression in particular cells, tissueS or organs of receptor-like kinase operably linked to a plant-operable a plant, of a nucleic acid encoding an LRR receptor-like promoter Sequence. kinase as defined in any of claims 15 to 17 operably linked 54. A method for enhancing overall growth and yield to a plant-operable promoter Sequence. comprising the expression in particular cells, tissueS or 42. A method Stimulating root meristem formation com organs of a plant, of a nucleic acid encoding an LRR prising the expression in particular cells, tissueS or organs of receptor-like kinase as defined in any of claims 15 to 17 a plant, of a nucleic acid encoding an LRR receptor-like operably linked to a plant operable promoter Sequence. kinase under the control of a transcriptional regulatory root 55. A method for enhancing overall growth and yield promoter as defined in any of claims 2 to 5. comprising the expression in particular cells, tissueS or 43. A method for stimulating root meristem formation organs of a plant, of a nucleic acid encoding an LRR comprising the expression in particular cells, tissueS or receptor-like kinase under the control of a transcriptional organs of a plant, of a nucleic acid encoding a polypeptide regulatory root promoter as defined in any of claims 2 to 5. as defined in any of claims 15 to 17 under the control of a 56. A method for enhancing overall growth and yield transcriptional regulatory root promoter as defined in any of comprising the expression in particular cells, tissueS or claims 2 to 5. organs of a plant, of a nucleic acid encoding a polypeptide 44. A method for root-meristem maintenance comprising as defined in any of claims 15 to 17 under the control of a the expression of a gene that influences the proliferation transcriptional regulatory root promoter as defined in any of and/or differentiation of the root meristem under the control claims 2 to 5. of a transcriptional regulatory root-Specific promoter as 57. A method for modifying cell fate and/or plant devel defined in any of claims 2 to 5. opment and/or plant morphology and/or plant biochemistry 45. A method for root-meristem maintenance comprising and/or plant physiology comprising the modification of the expression in particular cells, tissueS or organs of a plant, expression in particular cells, tissueS or organs of a plant, of of a nucleic acid encoding an LRR receptor-like kinase a nucleic acid encoding an LRR receptor-like kinase oper operably linked to a plant-operable promoter Sequence. ably linked to a plant-operable promoter Sequence. 58. A method for modifying cell fate and/or plant devel 46. A method for root-meristem maintenance comprising opment and/or plant morphology and/or plant biochemistry the expression in particular cells, tissueS or organs of a plant, and/or plant physiology comprising the modification of of a nucleic acid encoding an LRR receptor-like kinase as expression in particular cells, tissueS or organs of a plant, of defined in any of claims 15 to 17 operably linked to a a nucleic acid encoding an LRR receptor-like kinase as plant-operable promoter. defined in any of claims 15 to 17 operably linked to a 47. A method for root-meristem maintenance comprising plant-operable promoter Sequence. the expression in particular cells, tissueS or organs of a plant, 59. A method for modifying cell fate and/or plant devel of a nucleic acid encoding an LRR receptor-like kinase opment and/or plant morphology and/or plant biochemistry under the control of transcriptional regulatory root promoter and/or plant physiology comprising the modification of as defined in any of claims 2 to 5. expression in particular cells, tissueS or organs of a plant, of 48. A method for root-meristem maintenance comprising a nucleic acid encoding an LRR receptor-like kinase under the expression in particular cells, tissueS or organs of a plant, the control of a transcriptional regulatory root promoter as of a nucleic acid encoding a polypeptide as defined in any of defined in any of claims 2 to 5. claims 15 to 17 under the control of a transcriptional 60. A method for modifying cell fate and/or plant devel regulatory root promoter as defined in any of claims 2 to 5. opment and/or plant morphology and/or plant biochemistry 49. A method for enhancing root formation and/or root and/or plant physiology comprising the modification of growth comprising the expression in particular cells, tissues expression in particular cells, tissueS or organs of a plant, of US 2004/OO67506 A1 Apr. 8, 2004 58 a nucleic acid encoding a polypeptide as defined in any of interacting with said LRR receptor-like kinase (protein) claims 15 to 17 under the control of a transcriptional or an interacting protein obtainable by a method of regulatory root promoter as defined in any of claims 2 to 5. claim 65 or 66 are expressed, 61. A method to confer pathogen resistance to a plant comprising the expression in particular cells, tissueS or (b) interacting said compound with the complex formed organs of a plant, of a nucleic acid encoding an LRR by the expressed proteins as defined in a), receptor-like kinase operably linked to a plant-operable (c) detecting a second complex, wherein the presence of promoter Sequence. Said Second complex identifies a compound which 62. A method to confer pathogen resistance to a plant Specifically binds to one of Said polypeptides or Said comprising the expression in particular cells, tissueS or Second complex, and organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase as defined in any of claims 15 to 17 (d) identifying the compound. operably linked to a plant-operable promoter Sequence. 69. A method for identifying compounds or mixtures of 63. A method to confer pathogen resistance to a plant compounds which specifically bind to a polypeptide of any comprising the expression in particular cells, tissueS or of claims 15 to 17, comprising: organs of a plant, of a nucleic acid encoding an LRR receptor-like kinase under the control of a transcriptional (a) combining a polypeptide of any of claims 15 to 17 regulatory root promoter as defined in any of claims 2 to 5. with Said compound or mixtures of compounds under 64. A method to confer pathogen resistance to a plant conditions Suitable to allow complex formation, and, comprising the expression in particular cells, tissueS or organs of a plant, of a nucleic acid encoding a polypeptide (b) detecting complex formation, wherein the presence of as defined in any of claims 15 to 17 under the control of a a complex identifies a molecule which specifically transcriptional regulatory root promoter as defined in any of binds Said polypeptide. claims 2 to 5. 70. Use of a molecule identifiable by means of a method 65. A method for identifying and obtaining proteins of claim 68 or 69 as a plant growth regulator or herbicide. interacting with a (LRR receptor-like kinase) polypeptide of 71. A method for the production of a plant growth any of claims 15 to 17 comprising a Screening assay wherein regulator or herbicide composition comprising the Steps of a polypeptide of any of claims 15 to 17 is used or expressed. the method of claim 68 or 69 and formulating the com 66. A method according to claim 65 comprising a two pounds obtained from Said Steps in a Suitable form for the hybrid Screening System wherein a nucleic acid encoding a application in agriculture or plant cell or tissue culture polypeptide of any of claims 15 to 17 as a bait and a cDNA 72. Use of a nucleic acid molecule of any of claims 1 to library as prey are expressed. 7, a vector of claim 10 to 12, a polypeptide of claim 15 to 67. A method for modulating the interaction between an 17 or an antibody of claim 19 for modifying cell fate and/or LRR receptor-like kinase (protein) as defined in any of plant development and/or plant morphology and/or plant claims 15 to 17 and interacting proteins obtainable by a biochemistry and/or plant physiology. method according to claim 65 or 66. 73. Diagnostic composition comprising at least one 68. A method for identifying and obtaining compounds nucleic acid molecule of any of claims 1 to 7, Vector of claim interacting with an LRR receptor-like kinase (protein) com 10 to 12, polypeptide of claim 15 to 17 or antibody of claim prising the Steps of 19. (a) providing a two-hybrid Screening System wherein a polypeptide of any of claims 15 to 17 and a protein