Proc. Natl. Acad. Sci. USAVol. 81, pp. 1728-1732, March 1984Cell Biology

Nucleotide sequence of the tms genes of the pTiA6NC octopine Tiplasmid: Two gene products involved in plant tumorigenesis

(Agrobacterium tumefaciens)

HARRY KLEE*, ALICE MONTOYA*, FRANK HORODYSKIt, CONRAD LICHTENSTEIN*T, DAVID GARFINKEL*§,SHERYL FULLER*t, CARLOS FLORES*, JACQUES PESCHON*, EUGENE NESTER*, AND MILTON GORDONt*Department of Microbiology and Immunology and tDepartment of Biochemistry, University of Washington, Seattle, WA 98195

Communicated by Earl W. Davie, December 12, 1983

ABSTRACT The nucleotide sequence of the tumor mor-phology locus, tms, from pTiA6NC has been determined. Thesequence analysis indicates that each of two polyadenylylatedtranscripts encoded by this locus contains an open readingframe; the predicted transcript 1 gene product has a molecularsize of 83,769 daltons, and the predicted transcript 2 geneproduct, of 49,588 daltons. The precise start and stop positionsof the transcript 2 RNA have been mapped with S1 nuclease.Several insertion mutations have been constructed. One ofthese localizes the transcript 2 promoter within the 72 basepairs 5' to transcription initiation. Significant homology wasobserved between the protein encoded by transcript 1 and theadenine binding region of p-hydroxybenzoate hydroxylasefrom Pseudomonasfluorescens, suggesting that the transcript1 protein binds adenine either as substrate or cofactor.

Crown gall is a neoplastic disease induced by Agrobacteriumtumefaciens affecting most dicotyledonous plants (reviewedin refs. 1 and 2). Both the ability to transform plant cells andhost range of the bacteria reside on a large plasmid, the Tiplasmid. The bacteria transfer a specific portion of the Tiplasmid to the plant cell, where this transferred DNA (T-DNA) is stably integrated into plant nuclear DNA. Axeniccultures of crown gall tissue exhibit autonomous growth inthe absence of the phytohormones, auxin, and cytokinin. Inaddition, transformed tissue directs the synthesis of opines,derivatives of certain amino acids that Ti plasmid-containingstrains of A. tumefaciens can use as sole sources of carbonand nitrogen.Tumors induced by the octopine-type Ti plasmid,

pTiA6NC, or the closely related pTiB6S3 synthesize eightpolyadenylylated transcripts from genes entirely within theT-DNA (3, 4). The DNA sequences of two of these genes,neither essential for tumor formation, have been reported.The product of transcript 3 is responsible for synthesis ofoctopine by transformed plant tissue (5). The function of theprotein encoded by transcript 7 is unknown (6). Five addi-tional transcripts encoded from three distinct genetic loci ap-pear to play a role in tumor morphology (7). Mutations inthese loci result in synthesis of shoots (tms), synthesis ofroots (tmr), or enlarged tumors (tml). The phenotypes ofthese different transformed tissues can be correlated withalterations in normal cytokinin/auxin ratios of the tissue (8)and can be corrected by addition of exogenous phytohor-mones (9). The function of the transcript 2 protein appears tobe conversion of indole-3-acetamide to the auxin, indoleace-tic acid (J. Schroder, personal communication). It seemslikely that the other tumor morphology genes are also in-volved in phytohormone biosynthesis, although this conclu-sion is by no means certain. We have determined the nucleo-

A2101A2100 A2102 328

BamHIEcoRI

HindIII

344 334

7.6 10.481 0.951.2 1.1 7.3

2.0 10.421 0.70 12.61 u

Bgl II Pvu II

2 1 4

FIG. 1. Restriction endonuclease map of the tms region ofpTiA6NC. Numbering of fragments is based on sizes in kb. The po-sitions and directions of transcripts 1 and 2 as well as for the tmrgene product, transcript 4, are indicated by horizontal arrows. Theexact positions of initiation and termination of transcript 1 have notbeen determined. Positions of insertions resulting in a tms morphol-ogy (A2100, 328, and 344) or a wild-type morphology (A2101,A2102, and 334) are indicated by vertical arrows.

tide sequences of two morphology genes, those encodingtms transcripts 1 and 2, as well as the sites of transcriptioninitiation and termination for the latter gene. Both of theseloci are implicated in conferring auxin autonomy to trans-formed plant tissue. A mutation in either locus confers thetms phenotype on a tumor. Sequence analysis indicates thattranscripts 1 and 2 contain open reading frames (orfs) capa-ble of encoding proteins of 83,769 and 49,588 daltons, re-spectively.

MATERIALS AND METHODSMaterials. Restriction endonucleases were purchased

from New England BioLabs or Bethesda Research Labora-tories and used according to the manufacturers' specifica-tions. DNA polymerase I Klenow fragment was obtainedfrom New England BioLabs. S1 nuclease was from BethesdaResearch Laboratories. [a-32P]dNTPs were purchased fromNew England Nuclear, and nonradiolabeled nucleotideswere purchased from P-L Biochemicals. Isopropylthio-,8-D-galactoside and 5-bromo-4-chloro-indoyl B-D-galactosidewere from Bethesda Research Laboratories.

Culture Conditions. LB broth and agar were prepared asdescribed by Miller (10). Agrobacterium strains were grownon AB minimal medium (11).

Nucleic Acid Sequence Determination. All plasmid andphage replicative form DNAs were prepared by the method

Abbreviations: T-DNA, DNA transferred from the Ti plasmid toa plant cell; orf, open reading frame; bp, base pair(s); kb, kilo-base pair(s).tPresent address: Biotechnology Centre, Imperial College, LondonSW7 2AZ, England.§Present address: Biology Department, Massachusetts Institute ofTechnology, Cambridge, MA 02139.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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of Clewell and Helinski (12). The strategies used in the se- S1 Nuclease Mapping of Transcript 2. Unique single-quence determination are described elsewhere (13). Briefly, strand-labeled probes spanning the 5' and 3' termini of tran-100- to 300-base-pair (bp) fragments ofT-DNA were generat- script 2 RNA were prepared as described (13) by in vitro-ed by restriction endonuclease digestion or by sonication. primed synthesis of M13 cloned DNAs. Hybridizations andThese fragments were cloned into M13 mp7, mp8, or mp9 S1 nuclease digestions were carried out as described (13).(14) and screened by plaque hybridization (15); their se- Isolation and Characterization of Mutations. TnS insertionquences were determined by the dideoxy methods described mutations previously isolated in this laboratory (7), whichby Sanger et al. (16, 17). The results are shown in Fig. 1. are located in and around the tms locus, were accurately

Isolation of RNA From Crown Gall Tissue. The octopine mapped (within 10 bp) by restriction endonuclease diges-crown gall line A6S2 (18) was grown as a suspension in hor- tions. Additional mutations in and adjacent to transcript 2mone-free MS medium (19) and harvested in late logarithmic were made in the following way: a clone of BamHI fragmentphase. Total RNA was prepared as described by Taylor and 8 [the 7.6-kilobase-pair (kb) fragment in Fig. 1], in the Bgl IIPowell (20) and polyadenylylated RNA was isolated by oli- site of pRK290 (7), was partially digested with EcoRI. Ago(dT)-cellulose chromatography (21). gene encoding kanamycin resistance isolated from pUC4K

S1 108tgagcg gc gaggtggacc cgcatgaacatcatattaaagaaagc c caattgtcgc tttccattggcatgtcaacgaacagtatgttccccgattctcaacttasata

135 162 189 2169&aacggttgatgtggttatttatctacac^aaaacgaatcttttctaacaATGTCACCTTCACCTCTCCTTGATAACCAGTGCGATCATCTCCCAACCAAAATCGTG

METS.rAlaS.rProLLuLeuAspAsnGlnCusAsDHisLeuProThrLusMetVal243 270 297 324

GATCTCACAATCOTCGATAAQCGCGATGAATTQOACCGCAGGGTTTCCGATGCC TTCTTAGAACGAGAAOCTTCTACQQQAAOGACOATTACTCAAATCTCCACCGAGAspLeuThrMetValAspLysAlaAsp~luLeuAspArgArgValSerAspAlaPhoLou~luArgoluAlaSerArg6GyArgArgIeThr~ln~llSerThr~lu

351 378 405 432TGCAGCGCTGGGTTAGCTTGCAAAAGGCTGCCGCATGGTCGCTTCcCCCAGATCTCAGCTGGTGGAAAGGTAGCAGTTCTCTcC6CCITATATCTATATTGGCAbAAAACysSerAlacGlLeuAlaisgLysArgLeuAlaAspclyArgPheProclu~llSerAla~lycGlLysValAlaValLeuSerAlaTyrIleTgrIle~lyLysol u

459 48B6 513 54CATTCTGGGOCGGATACTTGAATCGAAACCTTGGCGCoCGCcAACAGTGAGTGGTCTCGTTGCCATCGACTTGCoACCATTTTGCATOGATTTCTCCGAAGCACAACTAIllLevuGlgArglleLeu~luSerLysProTrpAlaArgAlaThrValSercGyLeuValAlalleAspLeuAlaProPheCys"etAspPheSerGluAlaGlnLeu567 594 621 64E!

ATCCAAGCCCTGTTTTTGCTOAGCGGTAAAACATOTGCACCGATTGATCTTAGTCATTTCGTGGCCATTTCAATCTCTAAGACTOCCGGCCTTTCGAACCCTGCCAATCleGlnAlaLeuPheLeuLeuSerclyLusArgCysAlaProlleAsDLeuSer~isph*ValAlalloesr~loesrLgsThrAlaolyPh*ArgThrLeuProMet

675 702 729 756CCGCTGTACGAGAATGGCACGATGAAATGCOTTACCGGGTTTACCATAAcCCTTGAAGGGCCGCTGCCATTTGACATGGTAGCTTATGGTCOAAACCTGATGCTGAAGProLeuTyrpluAsnGlyThrletLyscysValThrGlyPheThrileThrLeu~lu~luAlaValProPheAspMetValAlaTyrQlyArgAsnL@uMetLeuLys

783 810 837 864GGTTCGGCAGGTTCCTTTCCAACAATCGACTTGCTCTACGACTACAGACCOTTTTTTGACCAATGTTCCGATACTGGACGCATCGGCTTCTTTCCGGAGGATOTTCCTGlySerAlaclySerPhoProThrIleAspLeuLeuTyrAspTyrArgPrDPhePheAspclnCysSerAspSerolyArglIelycPh&PheProcluAspValPro

891 918 945 972AAGCCGAAAGTGGCGQTCATTGGCGCTGGCATTTCCGGACTCGTQOTCQCAAACGAACTGCTTCATGCTGOGGTAGACGATGTTACAATATATOAAGCAAQTGATCQTLysProLysValAlaValIlleGIgAlaGlylloSer~lyLeuValVaIAlaAsncluLeuLeu~isAla~lyValAspAspValThrIeTyrcluAlaSerAspArg

999 1026 1053 1080CTTCGA6CCAA6CTTTCGTCACATCCTTTCAGOGAC6CTCCTAGTCTCGTGGCCCCAAATCCCCCCCATCCCATTTCCTCCTCCTCCATTCTCCTTCTTTTTCTTCCTCVal~lyGlyLysLauTrpSerHisAlaPheArgAspAlaProSerValValAlaGluset~lyAla~etArgPhoProProAlaAlaPheCysLeuPhePhoPheLeu

1107 1134 1161 1188GAGCGTTACGGCCTGTCTTCCATCAGGCCC4TTCCCAAATCCCGGCACAGCGCACACTTACTTGGTCTACCAAGCGCTCCAATACATGTGGAAAGCCCGGCAGCTGCCAGluArgTyr~lyLauSerSerMetArgProPheProAsnPro~lyThrValAspThrTyrL~uValTyrGln~lyValGlnTyreetTrpLysAla~lyGonLeuPro

1215 1242 1269 1296CCGAAGCTGTTCCATCGCGTTTACAACGGTTGGCGTGCGTTCTTGAAGGACOGTTTCTATGAGCGCAATATTGTGTTGGCTTCGCCTGTCGCTATTACTCAGGCCTTGProLysLeuPh@HisArgValTyrAsnolyTrpArgAlaPhoLeuLysAsp~lyPheTyr~luArgAspIleValLouAlaStrProValAlalloThr~lnAlaLeu

1323 1350 1377 1404AAATCAGGAGACATTAGGTGGGCTCATGACTCCTGGCAAATTT6GCTGAACCGTTTC6GGAGG4AGTCCTTCTCTTCA6GGAT6ACA6AGATCTTTCT0GGCACACATLysSerGlyAspIleArgTrpAlaHisAspSerTrplnIl1eTrpL~uAsnArgPhe~lyArgOluSerPheSerSerilgIleGluArg IePheLeu~lyThrHis

1431 1458 1485 1512CCTCCTGGTGGTGAAACATGGAGTTTTCCTCATGATTGGGACCTATTCAACCTAATCOGAATAGGATCTGGCGGCTTTGGTCCAGTTTTTGAAAGCGGGTTTAT'TGAGProProlGlyeluThrTrpS1rPheProHisAspTrpAspLeuPheLysL0u1otlyIlelySer~lyGlyPheiGlProValPh~luSer6lyPheIIe~lu

1539 1566 1593 1620ATCCTCCGCTT6GTCATCAACG6ATATGAAGAAAATCAGCOCATGTOcCCTGAAGGAATCTCAGAACTTCCACGTCGGATCGCATCTGAAGTOGTTAACGGTTGTTCTIleLeuArgLeuValIleAsn0lyTyrGluQ1uAsnGlnArgMetCysprooluolyI1e6Sr.luLeuProArgArgIllAlaSeriluVelValAsnQGlValSer

1647 1674 1701 1728GTGAGCCAGCGCATATGCCATGTTCAAGTCA6CGCGATTCAGAAGGAAAAGACAAAAATAAAGATAACCCTTAAGAGCGGGATATCTGAACTTTATGATAAGGTGGTGValSorGlnArgIleCysHisValGlnValArgAlall@G1nLysGluLysThrLysIleLysIleArgLeuLysSer6GlyleSer61uLeuTyrAspLysValVa1

1755 1782 1809 1836GTCACATCTGGACTCGCAAATATCCAACTCAGGCATTQCCTGACATGCGATACCAATATTTTTCAGOCACCAGTGAACCAAGCGGTTGATAACACCCATATGACAGGAValThrSer~lyLtuAlaAsnIleGlnLeuArgHis5CysLeuThrCysAspThrAsnIlePheG1nAlaProValAsn6lnAlaValAspAsn~ereisMetThrOIw

1863 1890 1917 1944TCGTCAAAACTCTTCCTGATGACTGAACGAAAATTCTGGTTAGACCATATCCTCCCGTCTTGTGTCCTCATGGACGGGATCGCAAAAGCAGTGTATTGCCTGGACTATSerSerLysLouPh*Leu~etThrGluArgLysPheTrpLeuAspHisIleLouProSerCysValLounetAspclyIleAlaLysAlaValTyrCysLeuAspTyr

1971 1998 2025 2052GAGCCGCAGGATCCGAATGGTAAAGGTCTAGTGCTCATCAGTTATACATOGGAGGACOACTCCCACAAGCTGTTGCGGCTCCCCGACAAAA AGAGCGATTATGTCTGGluProGlnAspProAsnGIMLysolyLeuValLou~leSerTyrThrTrpcluAspAspSerHisLysLeuLeuAlaValProAspLysLysGluArgLouCysLeu

2079 2106 2133 2160CTGCC6GACGCAATTTC6AGATCTTTCCCGCGCTTTGCCCAGCACCTATTTCCTGCCTGCGCTGATTACGACCAAAATGTTATTCAACATGATTGOCTTACAGACGAGLeuArgAspAlaIllSerArgSerPheProAlaPheAlaGlnHisLeuPheProAlaCysAlmAspTyrAsp~lnAsnVllIeGlnHisAspTrpLeuThrAspolu

2187 2214 2241 2268AATGCCGGGGGAGCTTTCAAACTCAACCG¢CGCTGGTGACATTTTTATTCTGAAGAACTTTTCTTTCAAGCACTGGACACGGCTAATGATACCGGAGTTTACTTGGCGAsnAla~lyIVAlaPheLysLeuAsnArgArg~ly~luAspPheTyrSer~lu~luL~uPhePhoGlnAlaLeuAspThrAlaAsnAspThr~lyV-lTyrLeuAla

2295 2322 2349 2376OGTTGCAGTTGTTCCTTCACAGGTGGATGGGTGGAGGGT2CTATTCA4ACCGCGT0TAAC0CC2TCT4T2CAATTATCCACAATT4T84AGGCATTTTGGCAAA6GGCGlyCysSerCysSerPh@Thr~ly~lyTrpValolu61yAlaIle~lnThrAlaCysAsnAlaValCysAlalleIleHisAsnCys~lyoly~leLeuAlaLys6ly

2403 2430, 2457 2484AATCCTCTCGAACACTCTTCCAAQAGATATAACTACCOCACTACAAATTAstctatggatcctgttacaagtattgc-agttttataaattccattattaatggaatcAsnProL*u0luHisSrTrpLysArgTyrAsnTyrArgThrArgAsn

2511 2538 2565 2592ttgattttaacactagctaaagagtttataatat ta gg tcgaat ga atttataaa tt

FIG. 2. Nucleotide sequence of transcript 1. The orf is shown in uppercase letters, and the amino acid sequence is shown below thenucleotide sequence.

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(22) and containing EcoRI sticky ends was ligated into eachof the EcoRI sites in the Bam 8 clone. After selection onkanamycin-containing medium, clones having the kanamy-cin-resistance gene inserted into either the left or right end ofthe 1.1-kb EcoRI fragment were identified. These mutationswere introduced into pTiA6NC by the marker-rescue tech-nique as described by Garfinkel et al. (7). Mutant strainswere tested for virulence on Kalanchoe diagremontiana andNicotiana tabacum as described (23).

RESULTSDNA Sequence Determination. Mutational analyses using a

variety of techniques have indicated that the tms locus inpTiA6NC covers approximately 5 kb (7, 24, 25). RNA blot-ting of polyadenylylated transcripts has shown that the locusactually encodes two RNAs; transcript 1 (2.7 kb) and tran-script 2 (1.75 kb) (26) (Fig. 1). The sequence of the DNA ofthe region covering these two transcripts has been deter-mined and is shown in Figs. 2 and 3. There are two orfs ex-

tending in opposite orientations out from the 0.42-kb HindIIIfragment and separated by 250 bp. The first orf (tmsl) en-codes a protein with a subunit molecular weight of 83,769and is consistent with the position and direction of transcrip-tion of transcript 1. Transcript 2 contains a second orf (tms2)encoding a protein with a subunit molecular weight of49,588.Amino Acid Sequence Homology. To obtain some insight

into the functions of the tms gene products, a search for ho-mology to known proteins was carried out using the proteinsequence database of the Atlas of Protein Sequence andStructure (27). Tmsl displayed significant homology to p-hydroxybenzoate hydroxylase from Pseudomonas fluores-cens. Specifically, residues 239-263 of tmsl showed a highdegree of identity to residues 5-29 of the hydroxylase (Fig.4). Residues 5-31 of the hydroxylase have been determinedby crystallographic analysis to form a pocket that binds theadenine moiety of FAD (28). It thus seems likely that tmslalso binds adenine in some form either as substrate or cofac-

-.72EcoRl

tgc c tcag tcattc gtatcacacqgc gtgattgc tgaattc ccaccaa tatggcgcaagc tggg ttcaagc ttggtatatttatttgy tc tgatgggtttgaaat

T r..CAACTCAGAGAGATrGTGGCCAT rACcTCTTAGCCCAAAGCCTAGAACACCTGAAACGGAAAGACTACTCCTGCTTACAACTAG1AGAAACTCTOATACOCGOTM#ETVal1Ala Il1eThrSerLeuh 1aGl nSerLeuGl uli sLeuLyJsArgLyJsAsp TyrSerCysLeuG luLeuVa lG luThrLeuIlleAl aArg109?TG>TGAAGCTGCAAAATCATTAAACGG5CrTr T5TGC TACAGAC TGGGATCGT TTGCGGCGAA5C GCCAAAAAAAT TGATCGCCATGGAAACGQCCGGAGTAGGTC TTTCCC yseuAaAlLpsSerLeuAsnAIaLeuLeuAIaTh rAsp TrpAspGlyLoukr gArgSerAl aLysLys IleAspArgHisG I yAsnAIaQ yV IOl LeuCs217CCCATTCCACTCTGTTTTAAGGCGAACATCGCTACCGGCCTATTTCCCACAAGCGCCGC TACGCCGGCGC TGATAAACCAC TTGCCAAAGATACCATCCCGCGTCGCACl y Il eProLeuCysPh eLy sAl aAsn I IeAl a~hrGIlyValPh eProrhrSerAl aAlTh rPr oAl aLeu I IeAsnHi sLeuProL ys I IeProSerArgVa lAla325 PvuIICAAAGAC TTTTTTCAGCTGGAGCAC TGCCCGGTGCC TCGGGAAATATGCATGAG TTATCGTTTrGGAATrTACAAGCAACAAC TATGC CA(:CGGGGCGOTGCGAAACCCOGl uArgLeuPh eSerAlaGl yAlaLeuProG l yAlaSerGI yAsnl~e tHi sGIluLeuSerPh eG I y I Ie'rh rSFrAsnAsn ryrA laThrG1yA laVa lArgAsnPro4 si3TGGAATCCAGATC TGATACCAGGGGGC TCAAGCGGTGGTG TGGC TGC TGCGGTAGC AAGCCGAT>TGATG TTAGGCGOCCATAGG'C AC C GATACCGCGTGCCATCTGTCGC

Proc. NatL. Acad Sci USA 81 (1984) 1731

239 263tols I VAL ALA VAL ILE GLY ALA GLY ILE SER GLY LEU VAL VAL ALA ASN GLU LEU LEU HIS ALA CLY VAL ASP ASP VAL

Hyjdroxyjlas* VAL ALA ILE ILE GLV ALAGLV PRO SER CLV LEU LEU LEU GLY GLN LEU LEU HIS LYS ALA CLY ILE ASP ASN VAL

5 29

FIG. 4. Amino acid homology between the transcript 1 gene product and P. fluorescens p-hydroxybenzoate hydroxylase. Conserved aminoacids are enclosed. Functionally similar amino acids are indicated by asterisks.

tor. No significant homology was found for the transcript 2gene product.

Transcript Characterization. The 5' end of transcript 2 wasprecisely mapped by S1 nuclease protection of radiolabeledDNA prepared by in vitro fill-in of an M13 clone spanningthis region. A 404-bp Pvu II/EcoRI fragment (Fig. 1) waspurified following primed radiolabeling of a single-strandedM13 template containing an insertion of the 1.1-kb EcoRIfragment. When the probe complementary to transcript 2was hybridized to polyadenylylated RNA prepared fromplant tumors, a fragment of 338 bp was observed after S1nuclease digestion (Fig. 5A). This fragment determines the 5'end of transcript 2 to be 16 bp 5' of the ATG translationinitiation codon. When a probe of opposite orientation wasused, no S1 nuclease-protected fragments were observed.The 3' end of transcript 2 was determined in the same

manner except that the labeled probe for protection was aBgl II/HindIII fragment. In this case, protected fragmentspresent in approximately equal amounts were observed at313 bp and 323 bp (Fig. 5B). Thus, this RNA appears to beheterogeneous, ending 118 and 128 nucleotides beyond thetermination codon (positions 1530 and 1540, respectively).

Mutational Analysis of the tas Region. Several TnS inser-tion mutations were previously mapped to the tms region (7).Since the DNA sequences of both the tms region and TnS(29) have been determined, it was possible to map the sites of

A 13

622-7-

309

-42

404_-

I

FIG. 5. S1 nuclease mapping of the initiation and terminationsites for transcript 2. (A) 5'-End mapping. The S1 nuclease-resistantfragment resulting from hybridization of the Pvu II/EcoRI fragment(Fig. 1) 32P-labeled in the coding strand with poly(A)+ A6S2 RNAwas sized on a 6% polyacrylamide urea gel (lane 1). As a control, theprobe was hybridized to yeast tRNA and the hybrid was digestedwith nuclease S1 (lane 2). The undigested Pvu II/EcoRI probe isalso shown (lane 3). (B) 3'-End mapping. The HindIII/Bgl II frag-ment was labeled in the coding strand (Fig. 1). The lanes are ar-ranged as in A: hybridization with tumor RNA (lane 1) and tRNA(lane 2) followed by S1 digestion, undigested HindIII/Bgl II probe(lane 3). The positions of Hpa II-digested pBR322 size markers areshown to the left of the gels.

insertion by restriction endonuclease digestion. Three muta-tions, 328, 334, and 344, were mapped in this way. Both 328and 344 were localized within the orf of transcript 1 (posi-tions 337 and 1934, respectively) and are tms mutants. Muta-tion 334 mapped 85 bp downstream from the tisi orf (posi-tion 2531) and is wild type.Because no transposon insertions in the 0.42-kb HindIII

fragment were available and this restriction fragment likelycontains the promoters for both tms transcripts, we madeadditional mutations by inserting an EcoRI fragment encod-ing kanamycin resistance into each of the two EcoRI siteslocated in the 7.6-kb BamHI fragment (Fig. 1). The leftwardinsertion, A2100, is within the orf of transcript 2 (position982) and results in a tms phenotype. The rightward insertionis located 72 bp 5' to the start of transcript 2 (position -72).Insertion of the kanamycin-resistance gene in either orienta-tion (A2101 and A2102) results in a wild-type phenotype.These insertions thus appear to separate the two tms genesas well as localize the entire promoter for transcript 2 withinthe 72 bp between the EcoRI site and the start of transcrip-tion.

Analysis of Nontranslated Regions. We have observed aconserved 9-nucleotide sequence, T-T-T-C-A-A-G-G-A, lo-cated 100-200 nucleotides upstream from the start of tran-scription in a number ofT-DNA genes including transcripts 3(octopine synthase), 4, and 7 from pTiA6NC as well as nopa-line synthase from the nopaline Ti plasmid, pTiT37 (13). Al-though the exact position of the start of transcription fortranscript 1 is not known, a sequence closely resembling theconsensus, T-G-T-C-A-A-A-G-A, appears 85 nucleotides up-stream from the start of translation. In contrast, transcript 2does not have anything resembling this conserved sequencein the proper orientation in the 5' nontranscribed region. Theupstream region of transcript 2 does contain a sequence ho-mologous to the "TATA" box observed in most eukaryoticgenes (30) as well as a sequence resembling the "CCAAT"box, usually located 5' to the TATA box (31).Both of the tms transcripts are polyadenylylated in plant

cells. A search of the sequences 3' to the orfs of these twotranscripts reveals sequences resembling the canonical poly-adenylylation sequence, A-A-T-A-A-A. Sequences with afive out of six base match appear in several places 3' to theorf in both transcripts. Two of these, T-A-T-A-A-A and A-A-T-A-A-T, occur immediately preceding the sites of termi-nation in transcript 2. There is also a sequence located mid-way between translational and transcriptional termination(position 1459) that is a perfect 8-bp palindrome separated byone nucleotide. Such hairpin structures have been implicat-ed in prokaryotic transcription termination but do not appearto be involved in eukaryotic termination (reviewed in ref.32).

DISCUSSIONA. tumefaciens containing mutations in the tms locus inducetumors that have a proliferation of shoots on many hostplants. These tumors have greatly reduced levels of auxinscompared with tumors induced by the wild-type strain (8).At least some of these mutants contain elevated cytokininlevels as well. Addition of exogenous auxins to tumor tissueresults in a return to the wild-type, unorganized phenotype

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(9). Thus there is a suggestion that the tms locus codes forsome product(s) that synthesizes or stabilizes auxins or acti-vates the plant's own auxin biosynthetic pathway. Indeed,recent evidence suggests that the function of the transcript 2protein in tumors is to convert indole-3-acetamide to thephytohormone indoleacetic acid (J. Schroder, personal com-munication).RNA blot analyses suggest that the tms loci of pTiA6NC

and closely related octopine Ti plasmids code for two a-amanatin-sensitive polyadenylylated transcripts (3, 4). Wehave shown that each of these transcripts contains an orf;transcript 1 can encode a protein of 83,769 daltons and tran-script 2 can encode a protein of 49,588 daltons. The predict-ed size of the transcript 2 gene product agrees with the dataof Schroder et al. (33), who observe proteins of 74,000,49,000, and 28,000 daltons in Escherichia coli minicells con-taining clones covering the tms region. The predicted size oftmsl is somewhat larger than observed; however, this maysimply reflect an aberrant electrophoretic mobility on Na-DodSO4/polyacrylamide gels. Alternatively, the 74,000-dal-ton protein may represent a processed or degraded form ofthe tmnsl protein. We observe no gene in the tms or the adja-cent tmr region corresponding to a 28,000-dalton protein.A significant homology between tmsl and the adenine-

binding domain of P. fluorescens p-hydroxybenzoate hy-droxylase suggests that tmsl uses adenine as either a sub-strate or a cofactor. Although no significant homology toother regions of the hydroxylase was found, it is interestingto note that the product of the hydroxylase, a diphenolicacid, enhances the growth effect of indoleacetic acid byblocking its decarboxylation (34).The DNA sequences of these and other T-DNA genes pro-

vide few clues about the origin of these genes. That is, arethey prokaryotic or eukaryotic? Codon usage for these twogenes reveals neither a prokaryotic nor a eukaryotic bias(data not shown). The observation that the genes are ex-pressed at low levels in both plant cells and E. coli minicellssuggests that these genes are somewhat adaptable but arenot entirely suited for either situation. They are, however,capable of expressing at high enough levels to bring abouttransformation of the plant cell. Quantitation of the levels oftranscripts 1 and 2 by dot blot hybridization indicates thateach of these mRNAs represents