Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Jan;7(1):59–67. doi: 10.1128/mcb.7.1.59

Functional domains of a T-DNA promoter active in crown gall tumors.

W B Bruce, W B Gurley
PMCID: PMC365041  PMID: 3031482

Abstract

Promoter domains required for transcriptional expression of the 780 gene of T-right (pTi15955) were identified by deletion mutagenesis. Accurate quantitation of transcriptional activity of a series of 5' and internal deletion mutants was achieved by using a double gene vector containing a reference 780 gene as an internal standard. Results of the 5' deletions delineated an activator element located between -440 and -229 base pairs (bp) from the start of transcription. Removal of this region resulted in a 100-fold decrease in promoter activity. Two relatively small internal deletion/substitution mutations at positions -74 to -76 and -98 to -112 reduced promoter activity to 38 and 42%, respectively. In most cases large-scale internal deletions (38 to 151 bp) occurring in various locations from positions -12 to -348 bp caused a significant loss in major promoter activity. However, three internal deletions starting at position -37 and extending upstream as far as -153 bp either had little effect on transcriptional activity or resulted in increased activity. Removal of the TATA motif drastically reduced promoter activity to less than 0.1% of the wild type. A minor start of transcription was detected 60 bases upstream from the major transcriptional start site. This minor promoter shares the same activator element as the major promoter for full activity. Deletion and insertion mutations downstream of the minor promoter TATA demonstrated the role of the TATA box in positioning the start of transcription.

Full text

PDF
59

Images in this article

62Image
on p.62
63Image
on p.63
65Image
on p.65

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
  2. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  3. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chilton M. D., Currier T. C., Farrand S. K., Bendich A. J., Gordon M. P., Nester E. W. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3672–3676. doi: 10.1073/pnas.71.9.3672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Church G. M., Ephrussi A., Gilbert W., Tonegawa S. Cell-type-specific contacts to immunoglobulin enhancers in nuclei. 1985 Feb 28-Mar 6Nature. 313(6005):798–801. doi: 10.1038/313798a0. [DOI] [PubMed] [Google Scholar]
  7. Cohen R. B., Sheffery M., Kim C. G. Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene. Mol Cell Biol. 1986 Mar;6(3):821–832. doi: 10.1128/mcb.6.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dierks P., van Ooyen A., Cochran M. D., Dobkin C., Reiser J., Weissmann C. Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells. Cell. 1983 Mar;32(3):695–706. doi: 10.1016/0092-8674(83)90055-7. [DOI] [PubMed] [Google Scholar]
  9. Dynan W. S., Tjian R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. 1985 Aug 29-Sep 4Nature. 316(6031):774–778. doi: 10.1038/316774a0. [DOI] [PubMed] [Google Scholar]
  10. Fluhr R., Kuhlemeier C., Nagy F., Chua N. H. Organ-specific and light-induced expression of plant genes. Science. 1986 May 30;232(4754):1106–1112. doi: 10.1126/science.232.4754.1106. [DOI] [PubMed] [Google Scholar]
  11. Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S., Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S. Expression of bacterial genes in plant cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4803–4807. doi: 10.1073/pnas.80.15.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fromm M., Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J Mol Appl Genet. 1982;1(5):457–481. [PubMed] [Google Scholar]
  13. Ghosh P. K., Lebowitz P., Frisque R. J., Gluzman Y. Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs. Proc Natl Acad Sci U S A. 1981 Jan;78(1):100–104. doi: 10.1073/pnas.78.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
  15. Gurley W. B., Czarnecka E., Nagao R. T., Key J. L. Upstream sequences required for efficient expression of a soybean heat shock gene. Mol Cell Biol. 1986 Feb;6(2):559–565. doi: 10.1128/mcb.6.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gurley W. B., Kemp J. D., Albert M. J., Sutton D. W., Callis J. Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2828–2832. doi: 10.1073/pnas.76.6.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  18. Kaulen Hildegard, Schell Jeff, Kreuzaler Fritz. Light-induced expression of the chimeric chalcone synthase-NPTII gene in tobacco cells. EMBO J. 1986 Jan;5(1):1–8. doi: 10.1002/j.1460-2075.1986.tb04169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klee H., Montoya A., Horodyski F., Lichtenstein C., Garfinkel D., Fuller S., Flores C., Peschon J., Nester E., Gordon M. Nucleotide sequence of the tms genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1728–1732. doi: 10.1073/pnas.81.6.1728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koncz C., De Greve H., André D., Deboeck F., Van Montagu M., Schell J. The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants. EMBO J. 1983;2(9):1597–1603. doi: 10.1002/j.1460-2075.1983.tb01630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kovacs B. J., Butterworth P. H. The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo. Nucleic Acids Res. 1986 Mar 25;14(6):2429–2442. doi: 10.1093/nar/14.6.2429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lis J. T., Simon J. A., Sutton C. A. New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene. Cell. 1983 Dec;35(2 Pt 1):403–410. doi: 10.1016/0092-8674(83)90173-3. [DOI] [PubMed] [Google Scholar]
  23. Matzke A. J., Chilton M. D. Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells. J Mol Appl Genet. 1981;1(1):39–49. [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  26. Merlo D. J., Nutter R. C., Montoya A. L., Garfinkel D. J., Drummond M. H., Chilton M. D., Gordon M. P., Nester E. W. The boundaries and copy numbers of Ti plasmid T-DNA vary in crown gall tumors. Mol Gen Genet. 1980;177(4):637–643. doi: 10.1007/BF00272674. [DOI] [PubMed] [Google Scholar]
  27. Murai N., Kemp J. D. Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1982 Jan;79(1):86–90. doi: 10.1073/pnas.79.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Myers R. M., Tilly K., Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science. 1986 May 2;232(4750):613–618. doi: 10.1126/science.3457470. [DOI] [PubMed] [Google Scholar]
  29. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  30. Odell J. T., Nagy F., Chua N. H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. 1985 Feb 28-Mar 6Nature. 313(6005):810–812. doi: 10.1038/313810a0. [DOI] [PubMed] [Google Scholar]
  31. Rothstein S. J., Jorgensen R. A., Postle K., Reznikoff W. S. The inverted repeats of Tn5 are functionally different. Cell. 1980 Mar;19(3):795–805. doi: 10.1016/s0092-8674(80)80055-9. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  33. Shaw C. H., Carter G. H., Watson M. D., Shaw C. H. A functional map of the nopaline synthase promoter. Nucleic Acids Res. 1984 Oct 25;12(20):7831–7846. doi: 10.1093/nar/12.20.7831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simpson J., Timko M. P., Cashmore A. R., Schell J., Montagu M. V., Herrera-Estrella L. Light-inducible and tissue-specific expression of a chimaeric gene under control of the 5'-flanking sequence of a pea chlorophyll a/b-binding protein gene. EMBO J. 1985 Nov;4(11):2723–2729. doi: 10.1002/j.1460-2075.1985.tb03995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomashow M. F., Nutter R., Montoya A. L., Gordon M. P., Nester E. W. Integration and organization of Ti plasmid sequences in crown gall tumors. Cell. 1980 Mar;19(3):729–739. doi: 10.1016/s0092-8674(80)80049-3. [DOI] [PubMed] [Google Scholar]
  36. Timko M. P., Kausch A. P., Castresana C., Fassler J., Herrera-Estrella L., Van den Broeck G., Van Montagu M., Schell J., Cashmore A. R. Light regulation of plant gene expression by an upstream enhancer-like element. Nature. 1985 Dec 12;318(6046):579–582. doi: 10.1038/318579a0. [DOI] [PubMed] [Google Scholar]
  37. Willmitzer L., Dhaese P., Schreier P. H., Schmalenbach W., Van Montagu M., Schell J. Size, location and polarity of T-DNA-encoded transcripts in nopaline crown gall tumors; common transcripts in octopine and nopaline tumors. Cell. 1983 Apr;32(4):1045–1056. doi: 10.1016/0092-8674(83)90289-1. [DOI] [PubMed] [Google Scholar]
  38. Willmitzer L., Otten L., Simons G., Schmalenbach W., Schröder J., Schröder G., Van Montagu M., de Vos G., Schell J. Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures. Mol Gen Genet. 1981;182(2):255–262. doi: 10.1007/BF00269667. [DOI] [PubMed] [Google Scholar]
  39. Winter J. A., Wright R. L., Gurley W. B. Map locations of five transcripts homologous to TR-DNA in tobacco and sunflower crown gall tumors. Nucleic Acids Res. 1984 Mar 12;12(5):2391–2406. doi: 10.1093/nar/12.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zambryski P., Depicker A., Kruger K., Goodman H. M. Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet. 1982;1(4):361–370. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES