Skip to main content
SpringerLink
Log in

Analysis of promoter activity from an α-zein gene 5′ flanking sequence in transient expression assays

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5′ flanking region of a 19 kDa α-zein gene. For these analyses, the zein promoter region was fused to the β-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5′ of the zein promoters. Zein upstream sequences enhanced transcription independently of the-189/-114 region. Although the-189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5′ of the zein promoter sequences. However, nucleotides-347 to-309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5′ of deletion mutants of the zein promoter also failed to produce GUS activity above background.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513–1523 (1979).

    PubMed  Google Scholar 

  2. Boston RS, Becwar MR, Ryan RD, Goldsbrough PB, Larkins BA, Hodges TK: Expression from heterologous promoters in electroporated carrot protoplasts. Plant Physiol 83: 742–746 (1987).

    Google Scholar 

  3. Boston RS, Kodrzycki R, Larkins BA: Transcriptional regulation of maize zein genes. In: Shannon LM, Chrispeels MJ (eds) Molecular Biology of Seed Storage Proteins and Lectins. Ninth Symposium in Plant Physiology, pp 117–126. University of California, River-side, CA (1986).

    Google Scholar 

  4. Boston BS, Larkins BA: Control of maize zein gene expression. In: Setlow J, Hollaender A (eds) Genetic Engineering, Principles and Methods, Vol 9, pp 61–74. Plenum Press, New York (1987).

    Google Scholar 

  5. Chourey PS, Zurawski DB: Callus formation from protoplasts of a maize cell culture. Theor Appl Genet 59: 341–344 (1981).

    Article  Google Scholar 

  6. Colot V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD: Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. EMBO J 6: 3559–3564 (1987).

    PubMed  Google Scholar 

  7. DeRose RT, Ma D-P, Kwon I-S, Hasnain SE, Klassy RC, Hall TC: Characterization of the kafirin gene family from sorghum reveals extensive homology with zein from maize. Plant Mol Biol 12: 245–256 (1989).

    Google Scholar 

  8. Ellis JG, Llewellyn DJ, Dennis ES, Peacock WJ: Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. EMBO J 6: 11–16 (1987).

    Google Scholar 

  9. Fang R-X, Nagy F, Sivasubramaniam S, Chua N-H: Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1: 141–150 (1989).

    Article  PubMed  Google Scholar 

  10. Grasser KD, Maier U-G, Haass MM, Felix G: Maize high mobility group proteins bind to CCAAT and TATA boxes of a zein gene promoter. J Biol Chem 265: 4185–4188 (1990).

    PubMed  Google Scholar 

  11. Jeang K-T, Khoury G: The mechanistic role of enhancer elements in eukaryotic transcription. Bioessays 8: 104–107 (1988).

    PubMed  Google Scholar 

  12. Jefferson RA: Assaying chimeric genes in plants, the GUS gene fusion system. Plant Mol Biol Report 5: 387–405 (1987).

    Google Scholar 

  13. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions, β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    PubMed  Google Scholar 

  14. Kamo KK, Chang KL, Lynn ME, Hodges TK: Embryonic callus formation from maize protoplasts. Planta 172: 245–251 (1987).

    Google Scholar 

  15. Kodrzycki R, Boston RS, Larkins BA: The opaque-2 mutation of maize differentially reduces zein gene transcription. Plant Cell 1: 105–114 (1989).

    Article  PubMed  Google Scholar 

  16. Krens FA, Molendijk L, Wullems GJ, Schilperoort RA: In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72–74 (1982).

    Google Scholar 

  17. Kriz AK, Boston RS, Larkins BA: Structural and transcriptional analysis of DNA sequences flanking genes that encode 19 kilodalton zeins. Mol Gen Genet 207: 90–98 (1987).

    PubMed  Google Scholar 

  18. Larkin PJ: Purification and viability determinations of plant protoplasts. Planta 128: 213–216 (1975).

    Google Scholar 

  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275 (1951).

    Google Scholar 

  20. Lyznik LA, Ryan RD, Ritchie SW, Hodges TK: Stable co-transformation of maize protoplasts with gusA and neo genes. Plant Mol Biol 13: 151–161 (189).

  21. Maier U-G, Brown JWS, Schmitz LM, Schwall M, Dietrich G, Feix G: Mapping of tissue-dependent and independent protein binding sites to 5′ upstream regions of a zein gene. Mol Gen Genet 212: 241–245 (1988).

    Google Scholar 

  22. Maier U-G, Brown JWS, Toloczyki C, Feix G: Binding of a nuclear factor to a conserved sequence in the 5′ flanking region of zein genes from maize. EMBO J 6: 17–22 (1987).

    Google Scholar 

  23. Maier U-G, Grasser KD, Haab MM, Feix G: Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize. Mol Gen Genet 221: 164–170 (1990).

    PubMed  Google Scholar 

  24. Maniatis T, Fritsch GF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

    Google Scholar 

  25. Marris C, Gallois P, Copley J, Kreis M: The 5′ flanking region of a barley B hordein gene controls tissue and developmental specific CAT expression in tobacco plants. Plant Mol Biol 10: 359–366 (1988).

    Google Scholar 

  26. Matzke AJM, Stoger EM, Schernthaner JP, Matzke MA: Deletion analysis of a zein gene promoter in transgenic tobacco plants. Plant Mol Biol 14: 323–332 (1990).

    PubMed  Google Scholar 

  27. Odell JT, Knowlton S, Lin W, Mauvais CJ: Properties of an inserted transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter. Plant Mol Biol 10: 263–272 (1988).

    Google Scholar 

  28. Roussell DL, Boston RS, Goldsbrough PB, Larkins BA: Deletion of DNA sequences flanking a 19 kilodalton zein gene reduces its transcriptional activity in heterologous plant tissues. Mol Gen Genet 211: 202–209 (1988).

    Article  PubMed  Google Scholar 

  29. Shotwell MA, Boyer SK, Chesnut RS, Larkins BA: Analysis of seed storage protein genes of oats. J Biol Chem 265: 9652–9658 (1990).

    PubMed  Google Scholar 

  30. Shotwell MA, Larkins BA: The biochemistry and molecular biology of seed storage proteins. In: Marcus A (ed) The Biochemistry of Plants, A Comprehensive Treatise, Vol 15, pp 296–345. Academic Press, New York (1989).

    Google Scholar 

  31. Thompson GA, Larkins BA: Structural elements regulating zein gene expression. Bioessays 10: 108–113 (1989).

    PubMed  Google Scholar 

  32. Widholm JM: The use of fluorescein diacetate and phenosafranin for determining viability of cultured plant cells. Stain Technol: 189–194 (1972).

Download references

Author information

Author notes

  1. Gary A. Thompson & Brian A. Larkins

    Present address: Department of Plant Sciences, University of Arizona, Forbes Hall, 8721, Tucson, AZ, USA

  2. Rebecca S. Boston

    Present address: Department of Botany, North Carolina State University, 27695-7612, Raleigh, NC, USA

Authors and Affiliations

  1. Department of Botany and Plant Pathology, Purdue University, 47907, West Lafayette, IN, USA

    Gary A. Thompson, Rebecca S. Boston, Leszek A. Lyznik, Thomas K. Hodges & Brian A. Larkins

Authors
  1. Gary A. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca S. Boston
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leszek A. Lyznik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas K. Hodges
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brian A. Larkins
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thompson, G.A., Boston, R.S., Lyznik, L.A. et al. Analysis of promoter activity from an α-zein gene 5′ flanking sequence in transient expression assays. Plant Mol Biol 15, 755–764 (1990). https://doi.org/10.1007/BF00016125

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00016125

Key words

Search

Navigation