Abstract
The complete nucleotide sequence of the transferred region (T-DNA) of an octopine tumor inducing (Ti) plasmid fromAgrobacterium tumefaciens (pTi15955) has been determined. A total of 24 595 nucleotides extending approximately 900 bases to either side of the outermost, T-DNA boundaries was sequenced. Computer analysis of the sequenced portion of the Ti plasmid revealed that recognition sites for 72 restriction endonucleases are present in the DNA sequence at least once; no site forEcoK exists in this DNA sequence. Two imperfect 24 base repeats border the T-DNA sequence; the left starts at position 909 and the right ends at position 23 782, giving the T-DNA region a total length, of 22 874 nucleotides. Another two similar 24 base repeats lie within T-DNA and divide it, into three distinct domains: T-left (TL-DNA) 13 175 bp of apparently eukaryotic origin; T-center (TC-DNA) 1816 bp of prokaryotic origin; and T-right (TR-DNA) 7 883 bp of eukaryotic origin. The T-DNA contains nine reported transcripts, however, 26 open reading frames longer than 300 bases that start with an ATG initiation codon were found. Fourteen open reading frames are bounded by putative eukaryotic promoters, ribosome binding sites, and poly(A) addition sites and occur only in TL-and TR-DNAs. No open reading frames showing eukaryotic promoter sequences are located within the TC-DNA.
Similar content being viewed by others
References
Ansorge W, Barker RF: System for DNA sequencing with resolution up to 600 base pairs. J Biochem Biophys Methods: in press, 1983.
Bendich AJ, McCarthy BJ: DNA comparisons among Barley, Oats, Rye and Wheat. Genetics 65:545–565, 1970.
Breathnach R, Chambon P: Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383, 1981.
Chilton M-D, Drummond MH, Merlo DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW: Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–274, 1977.
De Beuckeleer M, Lemmers M, De Vos G, Willmitzer L., Van Montagu M, Schell J: Further insight on the transferred DNA of octopine crown gall. Mol Gen Genet 183:283–288, 1981.
De Framond AJ, Barton KA, Chilton M-D.: Mini-Ti: new vector strategy for plant genetic engineering. Bio/Technology 1:262–269, 1983.
De Greve H, Decraemer H, Seurinck J., Van Montagu, M, Schell J: The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6S3. Plasmid 6:235–248, 1981.
De Greve H, Dhaese P, Seurinck J, Lemmers M, Van Montagu M, Schell J: Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1:499–511, 1983.
Dhaese P, DeGreve H, Gielen J, Seurinck J, Van Montagu M, Schell J: I dentification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO J 2:419–426, 1983.
Engler G, Depicker A, Maenhaut R, Villarroel-Mandiola R, Van Montagu M, Schell J: Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti-plasmid of Agrobacterium tumefaciens. J Mol Biol 152:183–208, 1981.
Fitzgerald M, Shenk T: The sequence 5′-AAUAAA-3′ forms part of the recognition site for polyadenylation of late SV40 mRNAs. Cell 24:251–260, 1981.
Garfinkel DJ, Nester EW: Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:731–743, 1980.
Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW: Genetic analysis of crow gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153, 1981.
Garoff H, Ansorge W: Improvements of DNA sequencing gels. Anal Biochem 115:450–457, 1981.
Hille J, Klassen I, Schilperoort RA: Construction and application of R prime plasmids, carrying different segments of an octopine Ti plasmid from Agrobacterium tumefaciens, for complementation of vir genes. Plasmid 7:107–118, 1982.
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180, 1983.
Holsters M, Silva B, Van Vliet F, Genetello C, DeBlock M, Dhase P, Depicker A, Inzé D, Villaroel R, VanMontagu M, Schell J: The functional organization of the A. tumefaciens Plasmid pTiC58. Plasmid 3:212–230, 1980
Hooykaas PJJ, Den Dulk-Ras H, Ooms G, Schilperoort RA: Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens. J Bacteriol 143:1295–1306, 1980.
Koekman BP, Ooms G, Klapwijk PM, Schilperoort RA: Genetic map of an octopine Ti-plasmid. Plasmid 2:347–357, 1979.
Kozak M: Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucl Acids Res 9:5233–5252, 1981.
Leemans J, Deblaere R, Willmitzer L, DeGreve H, Hernalsteens JP, Van Montagu M, Schell J: Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152, 1982.
Lemmers M, De Beuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens JP, Van Montagu M, Schell J: Internal organization, boundaries and integration of Tiplasmid DNA in nopaline crown gall tumors. J Mol Biol 144:353–376, 1980.
Lilley DMJ: The inverted repeat as a recognizable structual feature in supercoiled DNA molecules. Proc Natl Acad Sci USA 77:6468–6472, 1980.
Maxam AM, Gilbert W: Sequencing end-labeled DNA with base-specific chemical cleavages. In Grossman L, Moldave K (eds) Methods in enzymology Vol 65. Academic Press: New York, 1980, pp. 499–560.
McPherson JC, Nester EW, Gordon MP: Proteins encoded by Agrobacterium tumefaciens Ti plasmid DNA (T-DNA) in crow galltumors. Proc Natl Acad Sci USA 77:2666–2670, 1980.
Merlo DJ, Nutter RC, Montoya AL, Garfinkel DJ, Drummond MH, Chilton M-D, Gordon MP, Nester EW: The boundaries and copy numbers of Ti plasmid T-DNA vary in crown gall tumors. Mol Gen Genet 177:637–643, 1980.
Murai N, Kemp JD: T-DNA of p Til 5955 from Agrobacterium tumefaciens is transcribed into a minimum of seven polyadenylated RNAs in sunflower crown gall tumor. Nucl Acids Res 10:1679–1689 1982.
Ooms G, Hooykaas PJJ, Moolsnaar G, Schilperoort RA: Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA function. Gene 14:33–50, 1981.
Ooms G, Klapwijk PM, Poulis JA, Schilperoort RA: Characterization of Tn904 insertions in octopine Ti-plasmid mutants of Agrobacterium tumefaciens. J Bacteriol 144:82–91, 1980.
Panayotatos N, Wells RD: Cruciform structures in supercoiled DNA, Nature 289:466–470, 1981.
Rosenberg M, Court D: Regulatory sequences involved in the promotion and termination, of RNA transcription. Ann Rev Genet 13:319–353, 1979.
Schröder G, Klipp W, Hillebrand A Ehring R, Koncz C, Schröder J: The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria. EMBO J 2:403–409, 1983.
Sheikholeslam S, Lin B-C, Kado CI: Multiple-size plasmids in Agrobacterium radiobacter and A. tumefaciens. Phytopathology 69:54–58, 1979.
Shine J, Dalgarno L: The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346, 1974.
Simpson RB, O'Hara PJ, Kwok W. Montoya AL, Lichtenstein C, Gordon MP, Nester EW: DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA. Cell 29:1005–1014, 1982.
Smith GR, Kunes SM, Schultz DW, Taylor A, Triman KL: Structure of Chi hotspots of generalized recombination. Cell 24:429–436, 1981.
Thomashow MF, Nutter RC, Montoya AL, Gordon MP, Nester EW: Integration and organization of Ti-plasmid sequences in crown gall tumors. Cell 19:729–739, 1980.
Ursic D, Slightom JL, Kemp JD: Agrobacterium tumefaciens T-DNA integrates into multiple sites of the sunflower crown gall genome. Mol Gen Genet 190:494–503, 1983.
Willmitzer L, Dhaese P, Schreier PH, Schmalenbach W, Van Montagu M, Schell J: Size, location and polarity of T-DNA-encoded transcripts in octopine and nopaline tumors. Cell 32:1045–1056, 1983.
Yadav NS, Vanderleyden J, Bennett DR, Barnes WM, Chilton M-D: Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci USA 79:6322–6326, 1982.
Yang F, Montoya AL, Merlo DJ, Drummond MH, Chilton M-D, Nester EW, Gordon MP: Foreign DNA sequences in crown gall teratomas and their fate during the loss of the tumorous traits. Mol Gen Genet 177:707–714, 1980.
Zaenen I, Van Larebeke N, Teuchy H, Van Montagu M, Schell J: Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J Mol Biol 86:109–127, 1974.
Zambryski P, Depicker A, Kruger K, Goodman HM: Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1:361–370, 1982.
Author information
Authors and Affiliations
Additional information
The complete nucleotide sequence showing the positions of all 73 restriction enzymes and six possible reading frames is available upon request.
Rights and permissions
About this article
Cite this article
Barker, R.F., Idler, K.B., Thompson, D.V. et al. Nucleotide sequence of the T-DNA region from theA grobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2, 335–350 (1983). https://doi.org/10.1007/BF01578595
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01578595