Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1986 Dec;6(12):4486–4492. doi: 10.1128/mcb.6.12.4486

Isolation of tobacco DNA segments with plant promoter activity.

L M Herman, M C Van Montagu, A G Depicker
PMCID: PMC367232  PMID: 3540612

Abstract

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.

Full text

PDF
4486

Images in this article

4488Image
on p.4488
4489Image
on p.4489

Selected References

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

  1. An G., Friesen J. D. Plasmid vehicles for direct cloning of Escherichia coli promoters. J Bacteriol. 1979 Nov;140(2):400–407. doi: 10.1128/jb.140.2.400-407.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  3. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  4. 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. 1982;1(6):499–511. [PubMed] [Google Scholar]
  5. Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M., Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 1985 Jul 11;13(13):4777–4788. doi: 10.1093/nar/13.13.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dhaese P., Hussey C., Van Montagu M. Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage phi 105. Gene. 1984 Dec;32(1-2):181–194. doi: 10.1016/0378-1119(84)90046-5. [DOI] [PubMed] [Google Scholar]
  7. Fried M., Griffiths M., Davies B., Bjursell G., La Mantia G., Lania L. Isolation of cellular DNA sequences that allow expression of adjacent genes. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2117–2121. doi: 10.1073/pnas.80.8.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gelvin S. B., Karcher S. J., DiRita V. J. Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors. Nucleic Acids Res. 1983 Jan 11;11(1):159–174. doi: 10.1093/nar/11.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gruenbaum Y., Naveh-Many T., Cedar H., Razin A. Sequence specificity of methylation in higher plant DNA. Nature. 1981 Aug 27;292(5826):860–862. doi: 10.1038/292860a0. [DOI] [PubMed] [Google Scholar]
  10. Herrera-Estrella L., Block M. D., Messens E., Hernalsteens J. P., Montagu M. V., Schell J. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 1983;2(6):987–995. doi: 10.1002/j.1460-2075.1983.tb01532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herrera-Estrella L., Van den Broeck G., Maenhaut R., Van Montagu M., Schell J., Timko M., Cashmore A. Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature. 1984 Jul 12;310(5973):115–120. doi: 10.1038/310115a0. [DOI] [PubMed] [Google Scholar]
  12. Holsters M., Silva B., Van Vliet F., Genetello C., De Block M., Dhaese P., Depicker A., Inzé D., Engler G., Villarroel R. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid. 1980 Mar;3(2):212–230. doi: 10.1016/0147-619x(80)90110-9. [DOI] [PubMed] [Google Scholar]
  13. Jones J. D., Dunsmuir P., Bedbrook J. High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J. 1985 Oct;4(10):2411–2418. doi: 10.1002/j.1460-2075.1985.tb03949.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leemans J., Langenakens J., De Greve H., Deblaere R., Van Montagu M., Schell J. Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene. 1982 Oct;19(3):361–364. doi: 10.1016/0378-1119(82)90027-0. [DOI] [PubMed] [Google Scholar]
  15. Leemans J., Shaw C., Deblaere R., De Greve H., Hernalsteens J. P., Maes M., Van Montagu M., Schell J. Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet. 1981;1(2):149–164. [PubMed] [Google Scholar]
  16. Lemmers M., De Beuckeleer M., Holsters M., Zambryski P., Depicker A., Hernalsteens J. P., Van Montagu M., Schell J. Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline grown gall tumours. J Mol Biol. 1980 Dec 15;144(3):353–376. doi: 10.1016/0022-2836(80)90095-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Rao R. N., Rogers S. G. Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene. 1979 Sep;7(1):79–82. doi: 10.1016/0378-1119(79)90044-1. [DOI] [PubMed] [Google Scholar]
  19. Reiss B., Sprengel R., Will H., Schaller H. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene. 1984 Oct;30(1-3):211–217. doi: 10.1016/0378-1119(84)90122-7. [DOI] [PubMed] [Google Scholar]
  20. Scherer S., Mann C., Davis R. W. Reversion of a promoter deletion in yeast. Nature. 1982 Aug 26;298(5877):815–819. doi: 10.1038/298815a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Teeri T. H., Herrera-Estrella L., Depicker A., Van Montagu M., Palva E. T. Identification of plant promoters in situ by T-DNA-mediated transcriptional fusions to the npt-II gene. EMBO J. 1986 Aug;5(8):1755–1760. doi: 10.1002/j.1460-2075.1986.tb04423.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 1983;2(3):411–417. doi: 10.1002/j.1460-2075.1983.tb01438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Van Lijsebettens M., Inzé D., Schell J., Van Montagu M. Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens. J Mol Biol. 1986 Mar 20;188(2):129–145. doi: 10.1016/0022-2836(86)90299-8. [DOI] [PubMed] [Google Scholar]
  25. Van den Broeck G., Timko M. P., Kausch A. P., Cashmore A. R., Van Montagu M., Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. 1985 Jan 31-Feb 6Nature. 313(6001):358–363. doi: 10.1038/313358a0. [DOI] [PubMed] [Google Scholar]
  26. Williams D. M., Duvall E. J., Lovett P. S. Cloning restriction fragments that promote expression of a gene in Bacillus subtilis. J Bacteriol. 1981 Jun;146(3):1162–1165. doi: 10.1128/jb.146.3.1162-1165.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES