Summary
We have used deletion mutagenesis to analyze a TR-DNA promoter from the octopine-type Ti plasmid pTiB6806. The promoter for the gene encoding mannopine synthase (mas) was cloned upstream of the bacterial kanamycin-resistance gene neomycin phosphotransferase II (NPT II). Bal31 deletion mutagenesis was used to generate deletion derivatives of the mas/NPTII gene beginning 1353 bp upstream of the initiation of transcription and extending to 120 bp downstream from the mRNA start site. Deletions that left intact 318 bp upstream of transcription initiation had no detectable effect on the ability of tumors harboring the deletion to synthesize correctly initiated mRNA or to grow on the kanamycin analogue G418. Deletion to — 318 destroyed the ability of sunflower crown gall tumors to grow on G418 although low levels of the mas/NPTII transcript were detected in one tumor line. Deletions that left only 57 bp upstream of transcription initiation allowed neither growth on G418 nor detectable mas/NPTII synthesis, even though the CCAAT and TATAA homologies were intact. The mas promoter is functional in Agrobacterium tumefaciens and we present data concerning the effects of the Bal31 deletions on the growth of A. tumefaciens on kanamycin.
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References
Amasino RM, Powell ALT, Gordon MP (1985) Changes in T-DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line. Mol Gen Genet 197:437–446
An G (1986) Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol 81:86–91
An G, Ebert PR, Yi B-Y, Choi C-H (1986) Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells. Mol Gen Genet 203:245–250
Banerji J, Rusconi S, Schaffner W (1981) Expression of a globin gene is enhanced by remote SV40 DNA sequences. Cell 27:299–308
Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350
Birnboim HC, Doly J (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1519
Bomhoff G, Klapwijk PM, Kester HCM, Schilperoort RA, Hernalsteens JP, Schell J (1976) Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol Gen Genet 145:177–181
Chilton M-D, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672–3676
Comai L, Schilling-Cordaro C, Mergia A, Houck CM (1983) A new technique for genetic engineering of Agrobacterium Ti plasmid. Plasmid 10:21–30
De Greve J, Dhaese P, Seurinck J, Lemmers M, Van Montagu M, Schell J (1982) Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet 1:499–511
Dellaporta SJ (1983) A plant minipreparation: version II. Plant Mol Biol Rep 1:19–21
Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Ellis JG, Ryder MH, Tate ME (1984) Agrobacterium tumefacines TR-DNA encodes a pathway for agropine biosynthesis. Mol Gen Genet 195:466–473
Favolaro J, Treisman R, Kamen R (1980) Transcription maps of polyoma virus-specific RNA: analysis by two dimensional nuclease Sl gel mapping. Methods Enzymol 65:718–749
Fraenkel DG, Levisohn SR (1967) Glucose and gluconate metabolism in an Escherichia coli mutant lacking phosphoglucose isomerase. J Bacteriol 93:1571–1578
Fromm M, Berg P (1983) Simian virus 40 early- and late-region promoter functions are enhanced by the 72 base pair repeats inserted at distant locations and inverted orientations. Mol Cell Biol 3:991–999
Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW (1981) Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153
Gelvin SB, Gordon MP, Nester EW, Aronson AI (1981) Transcription of the Agrobacterium Ti plasmid in the bacterium and in crown gall tumors. Plasmid 6:17–29
Gelvin SB, Thomashow MF, McPherson JC, Gordon MP, Nester EW (1982) Sizes and map positions of several plasmid DNA encoded transcripts in octopine-type crown gall tumors. Proc Natl Acad Sci USA 79:76–80
Gelvin SB, Karcher SJ, Goldsbrough PB (1985) Use of a TR T-DNA promoter to express genes in plants and bacteria. Mol Gen Genet 199:240–248
Hepburn AG, Clarke LE, Pearson L, White J (1984) The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2:315–329
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–198
Janssens A, Engler G, Zambryski P, Van Montagu M (1984) The nopaline C 58 T-DNA is transcribed in Agrobacterium tumefaciens. Mol Gen Genet 195:341–350
Karcher SJ, DiRita VJ, Gelvin SB (1984) Transcript analysis of TR-DNA in octopine-type crown gall tumors. Mol Gen Genet 194:159–165
Klee H, Montoya A, Horodyski F, Lichtenstein C, Garfinkel D, Fuller S, Flores C, Peschon J, Nester E, Gordon M (1984) Nucleotide sequence of the tms genes of the pTiA 6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis. Proc Natl Acad Sci USA 81:1728–1732
Komro CT, DiRita VJ, Gelvin SB, Kemp JD (1985) Site-specific mutagenesis in the TR-DNA region of octopine-type Ti plasmids. Plant Mol Biol 4:253–263
Koncz C, De Greve H, Andre D, De Boeck F, Van Montagu M, Schell J (1983) The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants. EMBO J 2:1597–1603
Legerski RJ, Hodnett JL, Gray HB (1978) Extracellular nucleases of pseudomonas Bal 31 III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res 5:1445–1464
Lichtenstein C, Klee H, Montoya A, Garfinkel D, Fuller S, Flores C, Nester EW, Gordon M (1984) Nucleotide sequence and transcript mapping of the tmr gene of the pTiA 6NC octopine Ti-plasmid: a bacterial gene involved in plant tumorigenesis. J Mol Appl Genet 2:354–362
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Methods Enzymol 65:499–560
Messing J, Geraghty D, Heidecker G, Hu N-T, Kridl J, Rubenstein I (1982) Plant gene structure. In: Kosuge T, Meredith CP, Hollaender A (eds) Genetic engineering of plants: an agricultural perspective. Basic Life Sciences, vol 26. Plenum Press, New York London pp 211–227
Montoya AL, Chilton M-D, Gordon MP, Sciaky D, Nester EW (1977) Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes. J Bacteriol 129:101–107
Morrison DA (1979) Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol 68:326–331
Murai N, Kemp JD (1982) T-DNA of pTi-15955 from Agrobacterium tumefaciens is transcribed into a minimum of seven polyadenylated RNA's in a sunflower crown gall tumor. Nucleic Acids Res 10:1679–1689
Ooms G, Hooykaas PJJ, VanVeen RJM, VanBeelaen P, Regensburg-Tuink TJG, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15–29
Parish JH, Kirby KS (1966) Reagents which reduce interactions between ribosomal RNA and rapidly labelled RNA from rat liver. Biochim Biophys Acta 129:554–562
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schroder G, Waffenschmidt S, Weiler EW, Schroder J (1984) The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem 138:387–391
Shaw CH, Carter GH, Watson MD, Shaw CH (1984) A functional map of the nopaline synthase promoter. Nucleic Acids Res 20:7831–7846
Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW (1980) Integration and organization of Ti-plasmid sequences in crown gall tumors. Cell 19:729–739
Thomashow MF, Hugly S, Buchholz WG, Thomashow LS (1986) Molecular basis for the auxin-independent phenotype of crown gall tumor tissues. Science 231:616–618
Timko MP, Kausch AP, Castresana C, Fassler J, Herrera-Estrella L, Van Den Broeck G, Van Montagu M, Schell J (1985) Light regulation of plant gene expression by an enhancer-like element. Nature 318:579–582
Van Larebeke N, Engler G, Holsters M, Van Den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall inducing ability. Nature 252:169–170
Viera J, Messing J (1982) The pUC plasmids, an M13mp 7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Winter JA, Wright RL, Gurley WB (1984) Map location of five transcripts homologous to TR-DNA in tobacco and sunflower crown gall tumors. Nucleic Acids Res 12:2391–2406
Zenke M, Grundstrom T, Matther H, Wintzerith M, Schatz C, Wildeman A, Chambon P (1986) Multiple sequence motifs are involved in SV 40 enhancer function. EMBO J 5:387–397
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DiRita, V.J., Gelvin, S.B. Deletion analysis of the mannopine synthase gene promoter in sunflower crown gall tumors and Agrobacterium tumefaciens . Mol Gen Genet 207, 233–241 (1987). https://doi.org/10.1007/BF00331583
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DOI: https://doi.org/10.1007/BF00331583