Sequence analysis of an insertion element, IS1131, isolated from the nopaline-type Ti plasmid of Agrobacterium tumefaciens

Gene. ! 14 (1992) 229-233 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

229

GENE 06423

Sequence analysis of an insertion element, IS!131, isolated from the nopaline-type Ti plasmid of

Agrobacterium tumefaciens (Plant tumor; transposable element; recombinant DNA; regulatory elements; opine; sequence comparison; distribution)

Hiroetsu Wabiko

Laboratory of Molecular Genetics. Biotecbnology Institute. Akita Prefectural College of Agriculture. Ohgata. Akita 010-04 (Japan) Received by A. Nakazawa: 12 August 1991 Revised/Accepted: 14 December/24 December 1991 Received at publishers: 17 February 1992

SUMMARY Transferred DNA (T-DNA) from several nopaline-type Ti plasmids ofAgrobacterium tumefaciens was previously shown to be variable in size due to the insertion of extra DNA segments. We found an insertion sequence (named IS/131) in the T-DNA of the strain PO22 and determined the nucleotide (nt) sequence. IS1131 is 2773 bp long, contains four open reading frames, and is flanked by 12-bp perfect inverted repeats (IR). An 8-bp direct repeat was found immediately outside the IR, and represents a target site of integration. Although the ISII31 nt sequence showed a limited degree of similarity to those of the previously reported IS elements of A. tumefaciens and to the central region of T-DNA, the terminal IR of ISII31 were highly homologous (up to 83%) to those of IS66 and IS866, suggesting that these three IS elements are related to each other. A number of ISll31-related copies were found in several pathogenic, as well as nonpathogenic, nopaline-type strains from different plant sources, and were distributed on both chromosomes and plasmids.

INTRODUCTION A soil bacterium, A. tumefaciens, causes crown gall tumors on a variety of host plants. A part (T-DNA) of the tumor-inducing plasmid (Ti plasmid), harbored by the bacterium, is transferred and integrated into plant DNA

Correspondence to: Dr. H. Wabiko, Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural Collegeof Agriculture, 2-2 Minami, Ohgata, Akita 010-04 (Japan) Tel. (8 !- 185)45-2026; Fax (81- 185)45-2678. Abbreviations: A., Agrobacterium; aa, amino acid(s); bp, base pair(s); cpm, counts/min; A, deletion; EtdBr, ethidium bromide; IR, inverted repeat(s); IS, insertion sequence(s); kb, kilobase(s) or 1000bp; nt, nucleotide(s); ORF, open reading frame; SDS, sodium dodecyl sulfate; TDNA, transferred DNA.

upon infection (Van Larebeke et al., 1974; Chilton et al., 1977). T-DNA contains the genes required for tumorous growth of plant tissues (Akiyoshi et al., 1984; Thomashow et al., 1984). Compounds, termed opines, are synthesized in the transformed tumorous tissues, and they include nopaline, octopine, cucumopine and others (reviewed by Nester et al., 1984). As the opine synthesis genes are located on T-DNA, the Ti-plasmids are classified according to the opine type. We previously suggested that the polymorphic structure of the T-DNAs from nopaline-type Ti-plasmids could be explained by insertion of extra DNA segments into the middle region of T-DNA (Wabiko et al., 1991). Here, I report that one of these inserts (IS/131) exists as multiple copies and indeed displays a structure typical of prokaryotic IS elements.

230 68 bp upstream from the ORF3 start codon, although the corresponding ' - 3 5 ' sequence ( 5 ' - T T G A C A ) was not found. None of the other ORFs were accompanied by either of the two regulatory elements. Klee et al. (1984) reported that in at least five T-DNA genes, a conserved sequence of 5 ' - T T T C A A G G A is present 100-200 bp upstream of the transcription start point. A similar sequence, 5 ' - T T T C A T G G C , was found 361 bp upstream from the start codon of ORF2.

EXPERIMENTAL AND DISCUSSION (a) Nucleotide sequence of IS1131 The restriction map of a part of the T-DNA from the strain PO22, including IS1131 (previously called ISC; Wabiko et al., 1991) and the corresponding region of the well-studied T-DNA of the nopaline-type strain T37 is shown in Fig. 1, ISl131 occurred between S a c l I (S) and Cfol (C) sites of PO22 T-DNA, but was absent in T37 T-DNA, and the nt sequence of these regions was determined (Fig. 2). IS1131 consisted of 2773 nt, possessed 12bp complete inverted repeats at both ends, with an 8-bp duplication immediately outside the IR (Fig. 2a). The PO22 T-DNA sequence surrounding IS 1131 was identical to that of the corresponding region (including the 8-bp sequence) of T37 T-DNA (Fig. 2b). Apparently, the 8-bp sequence is a target site of integration. These features represent a typical transposable insertion element. IS1131 contained four ORFs each over 300 bp in length. The ORFI (306 bp), ORF2 (537 bp), and ORF3 (639 bp) were found in the forward orientation, while ORF4 (522 bp) was in the reversed orientation. Examination of the upstream region of the coding sequence revealed that ORFI and ORF2 were preceded by sequences 5 ' - A A G A G G and 5 ' - T G G A G G , respectively, which are similar to the ribosome-binding sites ( 5 ' - A G G AGG) in E. colt. A sequence 5 ' - T A T A T T similar to the prokaryotic transcriptional element, ' - 10' sequence (Pribnow box, 5'-TATAAT) was present at nt position 868, i.e.,

(b) Comparison of ISll31 with known nt and aa sequences One orientation of IS1131 (Fig. 2) showed 46-50% homology to I S 6 6 (Machida et al., 1984), IS866 (Bonnard et al., 1989), and IS426 (Vanderleyden et al., 1986), while in the other orientation, homology was 46-48 % to IS426, IS427 (De Meirsman et al., 1990), and central region of T-DNA from pTi15955 (Baker et al., 1983). The striking nt sequence similarity was found in the terminal inverted repeats (Fig. 3). At least 6 nt were identical in the pair-wise comparisons of I S l l 3 1 , IS66 and IS866. Particularly, in the comparison of IS1131 and 1566, 8 nt were identical in the 12-nt left-proximal end, and 10 nt were identical in the 12-nt right-proximal end. From these analyses, it was concluded that the I S l l 3 1 , IS66, and I S 8 6 6 show some relationship with each other. Comparison of the deduced aa sequence of the ORFs did not reveal significant homology to the sequences present in the SWISS-PROT protein data bank except that similarity was observed between aa 69-184 of I S l l 3 1 ORF3

(e) L.

:: f

".F

1if'aTT-I]I lkb

(b)

s,

,c

S

K

S

K

S

S

H

$

C

B B

,S C !1

B

C mbdmm

'C

mm Immmm

0.lie Fig. I. Physicalmap ofT37 T-DNAand IS1131.(a) The restriction map ofT37 T-DNA.The map was taken from Depickerct al. (1980). The right border (RB) and left border (LB) are shown. (b) Subclones of ISll31. A part ofT37 T-DNA is expanded and the corresponding region of PO22 T-DNA including ISII31 (open box) is shown. Restriction fragmentswere sub¢loned into pUCI3 or pBluescript II (Stratagene, La Jolla, CA) vectors, with E. colt JM83 {ara .4(lac-pro) tllistrA (~aSOdlacZdM15)}and XLI-Blue{supE44 I~sdR17 recAI endA ! g,vrA46 thi relA I lac [F'proAB ÷ lacl q lacZ.4M 15Tni0 (tetR)]} as their host strains. The probe is the 0,8-kb HindIII fragment from ISll31. Sequential deletions were made from the 1.4-kb Sacll fragmentof ISil31 by BAL 31 digestion to obtain overlapping clones for nt sequencing. B, BamHI; C, Cfol; E, EcoRI; H, HindII1, K, Kpnl; S, SacII. The size in kb is indicated in (a) and (b).

231 (a) 60

GGAGGGTTGG AACTCCCTTG CCACATCATC GCGTTCGGTA G A G C G T ~ ~ G A G CGTCCGGTGA GCTGTTTTTG CTGATCGGGC AAATCAGGCG ATGTTCTGGC ATTCGAACCA ORFI GCATTAGTGC TGACATTAAT ACCAGAACAA AGAGGTTTC~GCTCGCAT GGAGATCATG

[20

TCCGGTACCG AGCGCAGGCG CCGTGGTCGG ACGAGGCGAA CGTCAGGATA TTGGCGGAAC KpnI GTGATGAACC CGGTGCTCGC ATTGGTGAGG TGGCGCGCCG GCATGACGTT CATCCTGGCC

240

AGATCCGCTT GTGGCGAGGT CGTTCAACTA TGTGGATCGA CCCACGGTGT TCCTTCCAGT

360

GGAAATCATG GAGGAGGCTG GCGTAAGTCA GGCGTCTTCG ACGGTAACGA GGCCGGCGAT

420

180

300

ORFI-=-~ CGTCGAGATA TTGCTGCGGA ACGGTCGGTG CCTGAAGGTT CCTGCTGACG TTGAGTTGAA r..-.~ORF2 GCTGCTCGGT CCGCTGATCG CTTGCGTGGA GGCGGCATGA TCGGGCCATC GGGCAATGTA

480

AGGGTCTATC TGGCCTGCGG AGTGACCGAT ATGCGGCGTG GCATTGATGG CCTATCGGCG

600

CTGGTCGAGA CGGTCGTGAA GGAGGC~CCG GGCTCGGGCG CAATTTTCGG CTTCCGCGGA

660

540

Sac£l AAGCGCGCCG ACCGGATCAA GCTTCTCTGG TGGGACGGCC AGGGGTTCTG CCTGTTCTAC Hind~I AAGATTTTGG AGCGTGGATA CTTTCCCTGG CCGACAGCGA AAGAAGGTGT CGCGCACCTG

~20

ACGCAGGCGC AGCTTTCCAT bCTGGTTGAG GGGATTGATT GGCGCGCCCG GCATGGACGT

840

CCGCTCCTGG CCGAACAGGA TAAAAGCTAT ATTTT,TCAAG GGCATCCGAG GCTTAACTCT

900

780

ORF4~RF3 GGCGCGTCAG AGGCAAATCG GCTAGATTTG CGGATATGGA AACAGCGCCG CTGGACAGTC

960

AGGACGAACT CACTGCTTTG CGCGCACTGG TCGCCGAACA GGCGGCGAAG CTTGAGAGCC ORF2=--.~, HLndZIl AGGAAGCCGA GGTCATCAAG C~AGACTCCA TCATAGGGCT TCTTCGCGC~ CAACTGGAGC

[020

TTCTCCGACA TCGGCAGCAT GGCGCCTCTT CGGAAAAGAT CGACCGGAAG ATAGAGCAGT

[[40

TCGAACTGAT GCTGGAGGAG ATTGAGGCTT CCCGTGCCGA GGCTGAACTG CGCTCCGGGA

[200

AAACACCCTT GCCGGATTTG GACGACGCGC CGGACAAGCC GAAGCGCAAA CCATTGCCCG

1260

ATGGTCTCGC CACCGAAGAG CTGATCTATG CGGCTCCCTG TAATTGCCC~ ACCTGCGGTG

[320

GCACCTCGTT CCTGAAGGCG CCCGACAGGG TGGTTCAGGT GCTGGAACAC GTGCCGGCGT

[380

CGGTCAAGAT TGTCCGCCAT GTCGAGAAGC GTATGATCTG CAAGGAATGC GATACGACAG ORF4 ~P==M IGGCTGGCGA GATGCCGACC TTGCCGATCG AGCGCGCAAG CCCGGGCCTG GATTGCTCGC

[440

I080

[500

Sma~ CCATATCATG GTCTCCAAGT TCGATGATCA CATTCCGCTT TACCGTCTCT CAGAGATGTA

[560

ORF3 TGATCGGCTG GGAATAGACA TATCGCGCTC CGTGACTAGC TGGGTCGGCG TATCGCTTTG

[620

CTGACACCCA TCGTCTTGTT GATCAGGGCC CACATCGCCG CGCTTGACCG AATACATACG

[680

GACGATACCC CG~TCGATGT TCTCGACCGG ACGGGGCAAG ACAAAAACCG GCAGGGTCTG

[740

GGTCTAGTCC TTCGACGGCA GTGGCTATCA AGCCACCACT CCGGCAGCCA TCGCCTATTA

[800

CTACAGTCCT GATCGAAAGG GCACACATCC GGCTGACeAC CTGGCAAGCT TTAGCGGCGT HindlII CATGCATGCC GACGGTTATG GCGGCTACAG ACAACTCTAC GGCAACCAGA TCGTTGAGGC

1920

[860

GCCTGCATGG CGCATGTACG TCGCAAGTTC CATGACGTGA TCAAGCTGAA GCCATCGCCG

[980

ATCGCCGACG AAGCTCTGTC CATCGGCGCG CTCTACGATA TCGAGGATCG TATCCGCGGA

2040

Sacll TGTCGGCTGA TGAGCGTCGT ACCCTGCGCC AACACCACGC CAGACCCATT CTGGACGAGC

2[00

TGAAGACCTG GATCGAGGCG ACACTCTCAA CTTTGCCACA GAAGCAGAAG CTGCCGAGGA

2160

ATGCGATATG CGCTGTCTCG ATGGGCAGCC TTGAGTTTAC ATCGAC~ATG GCCGTGTCGA

2220

AATCGATAAC AACATAGCTG AGCGAGCGAT GCGTCCGCTG GGCCTCGGCA GAAAAAACTG

2280

GTTATTCGCA GGCTCGGACA AGGGCGGTGA GCGCATCGCC AACATCCTGA CCATCATCGA

2340

AACGGTCAAA CTGCACGGCC ATAATCCGGA GCTCTACCTG ACAGATGTCC TGACCCGGAT

2400

and aa 1-118 of IS66 ORF2 (33~o identity) and between aa 1-106 ofISll31 ORF3 and aa 27-120 ofiS866 ORF5 (2770 identity). (c) Distribution of IS!131 in Agrobacterium tumefaciens strains Distribution of ISll31 was examined among nopalinetype A. tumefaciens strains from several plant sources (Wabiko et al., 1989). IS1131 was included in a single 7.6kb EcoRI fragmeitt of PO22 T - D N A . Total and plasmid D N A ofA. tumefaciens were digested with EcoRI and the fragments were subjected to Southern hybridization analysis, using a 0.8-kb HindIII fragment of ISII31 as a probe (Fig. 1). The strain PO22 (from poplar) contained at least six fragments of plasmids hybridized to the probe (Fig. 4, lane 2). The same hybridization pattern was observed for the total D N A preparation, which was composed of chromosomal and plasmid D N A (Fig. 4, lane !), suggesting that ISll31-related copies were present only on plasmids. The various degrees of hybridization signals in the plasmid fraction of PO22 may reflect different levels &homology among the ISll31-related copies or differences in the extent of overlap between the EcoRI fragments and the probe. In strain PO31 (from poplar), the same hybri0ization pattern was observed in total and plasmid D N A except for a 12-kb fragment, which was of chromosomal origin (Fig. 4, lanes 3 and 4). Moreover, total D N A of PO31 showed the same pattern as that of PO22 except that PO31 lacked the T-DNA-borne 7.6-kb fragment. Hybridization was not observed in either total or plasmid D N A of the strain, PO201 (from poplar; Fig. 4, lanes 5 and 6). it was next examined whether ISII31 was present in the natural poplar crown gall from which PO22, PO31 and PO201 were isolated. ~'he gall D N A displayed the hybridization pattern identical to that of PO31 and PO201 TD N A and lacked IS1131 (data not shown), suggesting that strains possessing the PO31- and PO201-type T - D N A played a major role in tumorigenicity. The strain PO22 might have developed during propagation of bacterial cells in crown-gall tissues. Since the distribution of ISII31 in PO22 was similar to that in PO31, ISIi31 could have

Bae~lI CCAGGATCAC CCCAAAGACC GATTTGAAGA TCTGCTGCCC TGGAACTGGA TGCCAGCAAA

2460

AGCTCGATGC GAGGCCGCCT GATGGCTCGC TCGAGGTTCA TCTATACGCT CAGCCAAGTC

2520

GCCGGCATGA TCGGCGAAAA CCTCGAACTG ATCGAAGAAG TAACCGCAAA CCCGGACAAC

2580

ATCTCCGAGG GCGAACTGGT TTACGTCAGC GATGGCAGTG .AAGATGGCAC GAAGGTCTGA

26~0

CCGGGAATGG CATCGAAGAA C,'D:CAAAGCC TACTTGCCGA CATCAGGACG TGGGACGGC"

2700

GTATCCGCGA GTTCCTCATC GACAGCCAGT GCGATCCTGA AATGATCGAC CGCGTCATGG

2760

CCGATGAAAT GAAACGCGGC TCATAGCTTC CATCTCTC~A TACCCGAAAA CGTTTGCCG__.~G 282g ACGCTCAC~T TCAGC~%AT ACTAGCTGAT GGCTCGTCCT GGGACTGGAA AAGCGTCGGG

2880

GAGAGTTC

2888

(b) GGAGGGTTGG AACTCCCTTG CCACATCATC GCGTTCGGTA G A G C G T ~ T A CTAGCTGATG GUTCGTCCTG GGACTGGAAA AGCGECGGGG AGAGTTC

Fig. 2. Nucleotide sequence of IS~HI. (a) Sequence of IS1131 and its flanking nt sequence were determined by the dideoxy chain-termination method of Sanger et al. (1977). The 12-bp terminal inverted repeatz =re indicated by being underlined with straight arrows, and the 8-bp direct repeat corresponding to the target site is marked by open boxes. Open reading frames (ORFI, ORF2, and ORF3)in the forwardorientationare shown above the sequence, and ORF4 in the reversed orientation is shown below the sequence. The nt sequence is deposited in the G~nBankTM/EMBL data base with the accession No. 82888. (b)Sequence of the T37 T-DNA corresponding to the region surrounding ISil31. The 8-bp sequence that is identical to the target site of ISII31 in (a) is boxed.

232 3'

end

5'

GTGAGCGTCCGG

II III

IS 1131 .

.

.

.

.

.

CCGGACGCTCAC

III

IIIII III II

GTAAGCCCACGGTGAAGGCC

..........

. . . . . . . . .

GTGAGCGTCCGG

II

.

I S 66

GGCCTTCACCGGAGGCTTAC

IS 1131

......... CCGGACGCTCAC

III I

I III

GTATGCGCCGCCTCCATCCCATTGATT---

I I

IS 66

II

GTATGCGCCGCCTCCATCCCATTGATT-

II

IS866---AATCAATGGGACGGAGACGCCGCATAC

..........

GTAAGCCCACGGTGAAGGCC

III II I

end

GGCCTTCACCGGAGGCTTAC

II

I

I II III

- - 'rs866 - - - A A T C A A T G G G A C G G A G A C G C C G C A T A C

Fig. 3, Paired comparison of the terminal IR of IS/131, IS66 and IS866. Left (5' end) and right (3' end) proximal sequences of the individual terminal inverted repeats are aligned, Identical nt are shown by vertical lines.

P022 T P 1 2

P031 T P 3 4

P0201 T P 5 6

PyTE1 T P 7 8 ,

PyTS3 AKEIO T P T P 9 10 11 12

[

kb

-9.4

,,,,~t,

-6.6

eVa¢~

-4.4

I

T37 PyTSI T P T P 13 14 15 16

PyTS2 T P 17 18

PyTS4 T P 19 20 " ~=" kb 9,4

6,6 r :,~. 4,4

Fig. 4 Occurrence of IS//M-related sequences in A. tumel~lcie,s. Total DNA ofA. tumefacie,s was slightly sheared, treated with alkali, and plas. raids were purified b) CsCI EtdBr density-gradient centrifugation (Currier anG Nester, 1976). Total DNA ~as prepared by the same procedure except that the sheafing and alkali-denaturation steps were omitted. Total DNA ( I ~ug)and plasmid DNA (0.3 lig) were digested with Ec.RI, separated b) 0.8°0 agamse gel electrophorcsis, and transferred on to GeneSc~'en Plus n.~lon membranes [Du Pont). Southern hybridization was performed as described previously (Sambrook el al., 1989). The probe (Im~e in Fig. I ) ~as radk~acti~el.~labeh.'d using a random primer kit (Ne~ England Nuclear) and [z-~:P]dCTP to give a specific acti~it.~ of 3 x IO'cpm Fg. ,Approx. 5 x IfPcpm (for samples from PO22, PO31. 1%.T$3. I~.T$1. I~.T$2. and ~TS4). or 5 x IO'cpm (for samples from

translocated to the middle of T-DNA, leading to generation of the strain PO22 in crown-gall tissues. Two to four hybridization signals were observed in total DNA preparation of the pathogenic strains, PyTE! (from pear) and T37 (from walnut), even though the signals were weaker than those in PO22 or PO31 (Fig. 4, lanes 7 and 13). Plasmid DNA preparations of PyTE 1 and T37 showed no hybridization (Fig. 4, lanes 8 and 14), indicating that these copies were possibly present only on the chromosome of these strains. Two insertion elements IS426 and IS427 had been isolated from T37 (Vanderleyden et al., 1986; De Meirsman et al., 1990). Since the two IS displayed a little homology to ISll31 (see above), weak hybridization signals in T37 may represent IS426 and/or IS427. A pathogenic strain, PyTS3, and nonpathogenic strains, PyTSI, PyTS2, and PyTS4, obtained from pear, also contained ISil31 both on the chromosomes and the plasmids in PyTS3, PyTS2, and PyTS4 but only on the chromosome in PyTS I (Fig. 4, lanes 9, 10, 15-20). No hybridization was observed in the pathogenic strain AKEI0 (from apple; Fig. 4, lanes i ! and 12). These results indicated that many ! S i 131-related copies occurred in multiple locations of various nopaline-type strains irrespective of their pathogenicity and plant sources.

(d) Conclusions (!) We determined the nt sequence of a novel insertion element, IS/131, from the T-DNA ofA. tumefaciens strain, PO22. The terminal inverted repeats of ISll31 displayed a significant simiiarity to IS66 and [$866 ofA. tumefaciens. (2) The nt sequence of a few upstream regulatory regions PO201, PyTEh AKEI0. and T37) was used. Membranes were washed in 2 x SSC 0.5% SD$ at 65:C. T and P refer to total DNA and plasmid DNA. respectivel.~. The molecular sizes were derived from Hindlll digests of phage ).. Pathogenic strains are PO22, PO31. PO201. ~ T E I . ~ T $ 3 . .AKEIO (Wabiko el al.. 1989). T37 (Sciak.~ et al.. 1978). and nonpathogenic stratus arc I~.TSI. ~ T S 2 . I~.TS4 (Wabiko ctal.. 1989).

233 associated with several ORFs resembled those of several T-DNA genes reported to date. (3) ISll31-related sequences were found in multiple locations in many nopaline-type A. tumefaciens strains. ACKNOWLEDGEMENTS

I thank Dr. H. Sano, S. Youssefian and H. Ebinuma for helpful suggestions and critical reading of the manuscript, and Ms. K. Futada for preparation of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (63616513) from the Ministry of Education, Japan. REFERENCES Akiyoshi, D.E., Klee, H., Amasino, R.M., Nester, E.W. and Gordon, M.P.: T-DNA of Agrobaclerium mmefacie,s encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81 (1984) 59945998. Baker, R.F., Idler, K.B., Thompson, D.V. and Kemp, J.D.: Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid ptil5955. Plant Mol. Biol. 2 (1983) 335-350. Bonnard, G., Vincent, F. and Otten, L.: Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium mmefaciens. Plasmid 22 (1989) 70-81. Chilton, M.-D., Drummond, M.H., Merlo, J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W.: Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell ! ! (1977) 263-271. Currier, T.C. and Nester, E.W.: Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76 ( ! 976) 43 !-44 I. De Meirsman, C., Van Soom, C., Verreth, C., Van Gool, A. and Vanderleyden, J.: Nucleotide sequence analysis of IS427 and its target sites in Agrobacrerium tume.laciens T37. Plasmid 24 (1990) 227-234. Depicker, A., De Wilde, M., De Vos, G., De Vos, R., Van Montagu, M. and Schell, J.: Molecular cloning of overlapping segments of the no-

paline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Piasmid 3 (1980) 193-211. Klee, HJ., Montoya, A., Horodyski, F., Lichtenstein, C., Gaffinkel, D., Fuller, S., FIores, C., Peschon, J., Nester, E. and Gordon, M.: Nucleotide sequence of the uns genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis. Proc. Natl. Acad. Sci. USA 81 (1984) 1728-1732. Machida, Y., Sakurai, M., Kiyokawa, S., Ubasawa, A., Suzuki, Y. and Ikeda, J.-E." Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid. Proc. Natl. Acad. Sci USA 81 (1984) 7495-7499. Nester, E.W., Gordon, M.P., Amasino, R.M. and Yanofsky, M.F.: Crown gall: a molecular and physiological analysis. Annu. Rev. Plant Physiol. 35 (1984) 387-413. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 54635467. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sciaky, D., Montoya, A.L. and Chilton, M.-D.: Fingerprints of Agrobacterium Ti plasmids. Plasmid I (1978)238-253. Thomashow, L.S., Reeves, S. and Thomashow, M.F.: Crown gall ontogenesis: evidence that a T-DNA gene from the AgrobacteriumTi plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid. Proc. Natl. Acad. Sci. USA 81 (1984) 5071-5075. Vanderleyden, J., Desair, J., De Meirsman, C., Michiels, K., Van Gool, A.P., Chilton, M.-D. and Jen, G.C.: Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous varient of the Ti-plasmid pTiT37. Nucleic Acids Res. 14 (! 986) 6699-6709. Van Larebeke, N., Engler, G., Holsters, M., Van den EIsacker, S., Zaenen, l., Schilperoort, R.A. and Schell, J.: Large plasmid in Agrobacterium tumefaciens is essential for crown gall-inducing ability. Nature 252 ( ! 974) 169-170. Wabiko, H., Kagaya, M., Kodama, l., Masuda, K., Kodama, Y., Yamamoto, H., Shibano, Y. and Sano, H.: Isolation and characterization of diverse nopaline type Ti plasmids ofAgrobacterium tumefaciens from Japan. Arch. Microbiol. 152 (I 989) I 19- ! 24. Wabiko, H., Kagaya, M. and Sano, H.: Polymorphism of nopaline-type T-DNAs from Agrobacterium tumefaciens. Plasmid 25 (1991) 3-15.