Molecular and General Genetics MGG

, Volume 231, Issue 2, pp 226–232 | Cite as

Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco

  • Mechthild Rieping
  • Fritz Schöffl


The thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5′ flanking region was unable to direct transcription from the TATA box of a truncated ΔCaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression fivefold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooporatively with HSEs to increase the hs promoter activity.

Key words

CCAAT box Heat shock element Enhancer Transgenic tobacco 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amin J, Ananthan J, Voellmy R (1988) Key features of heat shock regulatory elements. Mol Cell Biol 8:3761–3769Google Scholar
  2. Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179Google Scholar
  3. Baumann G, Raschke E, Bevan M, Schöffl F (1987) Functional analysis of sequences required for transcriptional activation of a soybean heat shock gene in transgenic tobacco plants. EMBO J 6:1161–1166Google Scholar
  4. Bevan MW (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721Google Scholar
  5. Bienz M (1986) A CCAAT box confers cell-type-specific regulation on the Xenopus hsp70 gene in oocytes. Cell 46:1037–1042Google Scholar
  6. Bienz M, Pelham HRB (1986) Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell 45:753–760Google Scholar
  7. Bienz M, Pelham HRB (1987) Mechanisms of heat shock gene activation in higher eukaryotes. Adv Genet 24:31–72Google Scholar
  8. Clos J, Westwood JT, Becker PB, Wolson S, Lambert K, Wu C (1990) Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell 63:1085–1097Google Scholar
  9. Czarnecka E, Key JL, Gurley WB (1989) Regulatory domains of the Gm hsp17.5-E heat shock gene promoter of soybean. A mutational analysis. Mol Cell Biol 9:3457–3463Google Scholar
  10. Czarnecka E, Fox PC, Gurley WB (1990) In vitro interaction of nuclear proteins with the promoter of soybean heat shock gene Gmhsp17.3-E. Plant Physiol 94:935–943Google Scholar
  11. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version 11. Plant Mol Biol Rep 1:19–21Google Scholar
  12. Gurley WB, Czarnecka E, Nagao RT, Key JL (1986) Upstream sequences required for efficient expression of a soybean heat shock gene. Mol Cell Biol 6:559–565Google Scholar
  13. Helm KW, Abernethy RH (1990) Heat shock proteins and their mRNAs in dry and early imbibing embryos of wheat. Plant Physiol 93:1626–1633Google Scholar
  14. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231Google Scholar
  15. Jakobsen BK, Pelham HRB (1991) A conserved heptapeptide restrains the activity of yeast heat shock transcription factor. EMBO J 10:369–375Google Scholar
  16. Jensen EQ, Marcker KA, Schell J, de Bruijn FJ (1988) Interaction of nodule specific, trans-acting factor with distinct DNA elements in the soybean leghaemoglobin lbc 3 5′ upstream region. EMBO J 7:1265–1271Google Scholar
  17. Jofuku KD, Okamuro JK, Goldberg RB (1987) Interaction of an embryo DNA binding protein with a soybean lectin gene upstream region. Nature 328:734–737Google Scholar
  18. Key JL, Lin CY, Chen YM (1981) Heat shock proteins of higher plants. Proc Natl Acad Sci USA 78:3526–3530Google Scholar
  19. Kingston RE, Schütz TJ, Larin Z (1987) Heat-inducible human factor that binds to a human hsp70 promoter. Mol Cell Biol 7:1530–1534Google Scholar
  20. Morgan WD, Williams GT, Morimoto RJ, Greene J, Kingston RE, Tjian R (1987) Two transcriptional activators, CCAATbox-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter. Mol Cell Biol 7:1129–1138Google Scholar
  21. Morimoto RI, Tissières A, Georgopoulos C (1990) Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  22. Neumann D, Never L, Parthier B, Rieger R, Scharf KD, Wollgiehn R, zur Nieden U (1989) Heat shock and other stress response systems of plants. Biol Zentbl 108:1–156Google Scholar
  23. Nover L (1987) Expression of heat shock genes in homologous and heterologous systems. Enzyme Microbiol Technol 9:130–144Google Scholar
  24. Parker CS, Topol J (1984) A Drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp70 gene. Cell 37:273–283Google Scholar
  25. Pedersen TJ, Arwood LJ, Spiker S, Guiltinan MJ, Thompson F (1991) High mobility group chromosomal proteins bind to ATrich tracts flanking plant genes. Plant Mol Biol 16:95–104Google Scholar
  26. Pelham HRB (1982) A regulatory upstream promoter element in the Drosophila hsp70 heat shock gene. Cell 30:517–528Google Scholar
  27. Perisic O, Xiao H, Lis JT (1989) Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 by recognition unit. Cell 59:797–806Google Scholar
  28. Raschke E, Baumann G, Schöffl F (1988) Nucleotide sequence analysis of soybean small heat shock protein genes belonging to two different multigene families. J Mol Biol 199:549–557Google Scholar
  29. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  30. Scharf KD, Rose S, Zott W, Schoffl F, Never L (1990) Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF. EMBO J 9:4495–4501Google Scholar
  31. Schöffl F, Baumann G (1985) Thermo-induced transcripts of a soybean heat shock gene after transfer into sunflower using a Ti plasmid vector. EMBO J 4:1119–1124Google Scholar
  32. Schöffl F, Baumann G, Raschke E, Bevan MW (1986) The expression of heat shock genes in higher plants. Philos Trans R Soc Lend [Biol] 314:453–468Google Scholar
  33. Schöffl F, Baumann G, Raschke E (1988) The expression of heat shock genes. A model for environmental stress response. In: Verma DPS, Goldberg RB (eds) Plant Gene Research — Temporal and Spatial Regulation of Plant Genes. Springer Verlag, Wien New York, pp 253–273Google Scholar
  34. Schöffl F, Rieping M, Baumann G, Bevan MW, Angermüller S (1989) The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco. Mol Gen Genet 217:246–253Google Scholar
  35. Schöffl F, Rieping M, Raschke E (1990) Functional analysis of sequences regulating the expression of heat shock genes in transgenic plants. In: Lycett GW, Grierson D (eds) Genetic Engineering of Crop Plants. Butterworth, London pp 79–94Google Scholar
  36. Schöffl F, Rieping M, Severin K (1991a) The induction of the heat shock response: activation and expression of chimaeric heat shock genes in transgenic plants. In: Herrmann RG (ed) Plant Molecular Biology. NATO-ASI Series. Plenum Press, New York, pp 685–694Google Scholar
  37. Schöffl F, Diedring V, Kliem M, Rieping M, Schröder G, Severin K (1991 b) The heat shock response in transgenic plants: the use of chimaeric heat shock genes. In: Wray JL (ed) Biochemistry and molecular biology of inducible enzymes and proteins in higher plants. Cambridge University Press, Cambridge, in pressGoogle Scholar
  38. Sorger PK, Lewis MJ, Pelham HRB (1987) Heat shock factor is regulated differently in yeast and HeLa cells. Nature 329:81–84Google Scholar
  39. Verwoerd TC, Dekker BMM, Hockema A (1989) A small-scale procedure for the rapid isolation of plant RNA. Nucleic Acids Res 17:2362Google Scholar
  40. Vieira J, Messing J (1982) The pUC plasmid, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar
  41. Vierling E, Sun A (1987) Developmental expression of heat shock proteins in higher plants. In: Cerry J (ed) Environmental Stress in Plants. Biochemical and Physiological Mechanisms associated with Environmental Stress Tolerance in Plants. Springer Verlag, New York, pp 343–354Google Scholar
  42. Williams GT, Morimoto RJ (1990) Maximal stress-induced transcription from the human hsp70 promoter requires interactions with the basal promoter elements independent of rotational alignment. Mot Cell Biol 10:3125–3136Google Scholar
  43. Wing D, Koncz C, Schell J (1989) Conserved function in Nicotiana tabacum of a single Drosophila hsp70 promoter heat shock element when fused to a minimal T-DNA promoter. Mot Gen Genet 219:9–16Google Scholar
  44. Wu BJ, Kingston RE, Morimoto RJ (1986) Human hsp70 promoter contains at least two distinct regulatory domains. Proc Natl Acad Sci USA 83:629–633Google Scholar
  45. Wu C (1984) Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin. Nature 311:81–84Google Scholar
  46. Xiao H, Lis JT (1988) Germline transformation used to define key features of heat shock response elements. Science 239:1139–1142Google Scholar
  47. Xiao H, Perisic O, Lis JT (1991) Cooperative binding of Drosophila heat shock factor to arrays of a conserved 5 bp unit. Cell 64:585–593Google Scholar
  48. Zimarino V, Wu C (1987) Induction of sequence-specific binding of Drosophila heat shock activator protein without protein syn thesis. Nature 327:727–730Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Mechthild Rieping
    • 1
  • Fritz Schöffl
    • 1
  1. 1.Department of GeneticsUniversity of TübingenTübingenGermany

Personalised recommendations