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Interaction of seed nuclear proteins with transcriptionally-enhancing regions of the pea (Pisum sativum L.) legA gene promoter

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Abstract

An 873-basepairs promoter fragment (-833 to +40), of the legA (legumin seed storage protein) gene of pea is known to be fully functional in transgenic plants. This fragment has been enzymically cleaved, and the products examined for the ability to interact specifically with nuclear proteins. Use of DNA-binding and mobility-shift assays has shown that promoter sequences between -316 and +40 do not form stable complexes with seed nuclear extracts. Fragments from -549 to -316 and -833 to -582, however, did interact strongly with seed, but not leaf, nuclear proteins. Each probe reacted similarly to form three distinct and stable complexes, although only the complex with least mobility appeared to be specific when challenged with competitor DNA fragments. Competitor studies also indicate that a single factor (designated LABF1) forms these specific low-mobility complexes with both probes. Fractionation of seed nuclear proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by elution and renaturation, shows that LABF1 activity resides in the 84 000- to 116 000-Mr size range of polypeptides. The tissue-specific activity of LABF1 is temporally correlated with legumin gene expression, a relationship consistent with suggestions that this factor may act as a transcriptional enhancer.

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Abbreviations

bp:

basepairs

DAA:

days after anthesis

EMSA:

electrophoretic mobility-shift assay

poly(dI.dC):

polydeoxyinosinic-deoxycytidylic acid

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  1. Allen, R.D., Bernier, F., Lessard, P.A., Beachy, R.N. (1989) Nuclear factors interact with a soybean β-conglycinin enhancer. Plant Cell 1, 623–631

  2. Baeurle, P.A., Baltimore, D. (1988) Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kB transcription factor. Cell 53, 211–217

  3. Bown, D., Ellis, T.H.N., Gatehouse, J.A. (1988) The sequence of a gene encoding convicilin from pea (Pisum sativum L.) shows that convicilin differs from vicilin by an insertion near the N-terminus. Biochem. J. 251, 717–726

  4. Bustos, M.M., Guiltinan, M.J., Jordano, J., Begum, D., Kalkan, F.A., Hall, T.C. (1989) Regulation of β-glucuronidase expression in transgenic tobacco plants by an A/T rich, cis-acting sequence found upstream of a french bean β-phaseolin gene. Plant Cell 1, 839–853

  5. Chen, Z-L., Pan, N-S., Beachy, R.N. (1988) A DNA sequence element that confers seed-specific enhancement to a constitutive promoter. EMBO J. 7, 297–302

  6. Clark, L., Pollock, R.M., Hay, R.T. (1988) Identification and purification of EBP1: A HeLa cell protein that binds to a region overlapping the “core” of the SV40 enhancer. Genes Devel. 2, 991–1002

  7. Cordes, S., Deikman, J., Margossian, L.J., Fischer, R.L. (1989) Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato. Plant Cell 1, 1025–1034

  8. Dickinson, C.D., Evans, R.P., Nielsen, N.C. (1988) RY repeats are conserved in the 5-flanking regions of legume seed protein genes. Nucleic Acids Res. 16, 371

  9. Dynan, W.S., Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. Nature 316, 774–778

  10. Evans, I.M., Gatehouse, J.A., Croy, R.R.D., Boulter, D. (1984) Regulation of the transcription of storage protein mRNA in nuclei isolated from developing pea (Pisum sativum L.) cotyledons. Planta 160, 559–568

  11. Garner, M.M., Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Applications to components of the E. coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060

  12. Gatehouse, J.A., Evans, I.M., Croy, R.R.D., Boulter, D. (1986) Differential expression of genes during legume seed development. Philos. Trans. R. Soc. London Ser.B 314, 367–384

  13. Green, P.J., Kay, S.A., Chua, N-H. (1987) Sequence specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcs-3A gene. EMBO J. 6, 2543–2549

  14. Holdsworth, M.J., Laties, G.G. (1989) Site-specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding. Planta 179, 17–23

  15. Jofuku, K.D., Okamuro, J.K., Goldberg, R.B. (1987) Interaction of an embryo DNA binding protein with a soybean lectin gene upstream region. Nature 328, 734–737

  16. Johnson, P.F., McKnight, S.L. (1989) Eukaryotic transcriptional regulatory proteins. Annu. Rev. Biochem. 58, 799–839

  17. Jordano, J., Almoguerra, C., Thomas, T.L. (1989) A sunflower helianthinin gene upstream sequence ensemble contains an enhancer and sites of nuclear protein interaction. Plant Cell 1, 855–866

  18. Kageyama, R., Pastan, I. (1989) Molecular cloning and characterization of a human DNA-binding factor that represses transcription. Cell 59, 815–825

  19. Kuwabara, M.D., Sigman, D.S. (1987) Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry 26, 7234–7238

  20. Lycett, G.W., Croy, R.R.D., Shirsat, A.H., Richards, D.M., Boulter, D. (1985) The 5-flanking regions of three pea legumin genes: comparison of the DNA sequences. Nucleic Acids Res. 13, 6733–6743

  21. Maier, U.G., Brown, J.W.S., Toloczycki, C., Feix, G. (1987) Binding of a nuclear factor to a consensus sequence in the 5′-flanking region of zein genes from maize. EMBO J. 6, 17–22

  22. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA

  23. Ptashne, M. (1986) Gene regulation by proteins acting nearby and at a distance. Nature 322, 697–701

  24. Riggs, C.D., Voelker, T.A., Chrispeels, M.J. (1989) Cotyledon nuclear proteins bind to DNA fragments harbouring regulatory elements of phytohemagglutinin genes. Plant Cell 1, 609–621

  25. Roussell, D.L., Boston, R.S., Goldsbrough, P.B., Larkins, B.A. (1988) Deletion of DNA sequences flanking an Mr 19 000 zein gene reduces its transcriptional activity in heterologous plant tissues. Mol. Gen. Genet. 211, 202–209

  26. Shirsat, A., Wilford, N., Croy, R., Boulter, D. (1989) Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol. Gen. Genet. 215, 326–331

  27. Shirsat, A., Meakin, P.J., Gatehouse, J.A. (1990) Sequences 5 to the conserved 28bp box element regulate the expression of pea seed storage protein gene legA. Plant Mol. Biol. 15, 685–693

  28. Singh, H., Sen, R., Baltimore, D., Sharp, P.A. (1986) A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature 319, 154–158

  29. Slightom, J.L., Sun, S.M., Hall, T.C. (1983) Complete nucleotide sequence of a french bean storage protein gene: Phaseolin. Proc. Natl. Acad. Sci. USA 80, 1897–1901

  30. Thompson, A.J., Evans, I.M., Boulter, D., Croy, R.R.D., Gatehouse, J.A. (1989) Transcriptional and post-transcriptional regulation of seed storage-protein gene expression in pea (Pisum sativum L.). Planta 179, 279–287

  31. Von der Haar, R.A., Allen, R.D., Cohen, E.A., Nessler, C.L., Thomas, T.L. (1988) Organisation of the sunflower 11S storage protein gene family. Gene 74, 433–443

  32. Yamamota, K.K., Gonzalez, G.A., Biggs, W.H., Montiminy, M.R. (1988) Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334, 494–498

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Additional information

The authors would like to thank Mr. D. Bown and Dr. A. Shirsat for providing subclones and also helpful discussion. Financial support provided by the Agricultural and Food Research Council (grant number PG12/28) is gratefully acknowledged.

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Meakin, P.J., Gatehouse, J.A. Interaction of seed nuclear proteins with transcriptionally-enhancing regions of the pea (Pisum sativum L.) legA gene promoter. Planta 183, 471–477 (1991). https://doi.org/10.1007/BF00194266

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Key words

  • DNA-binding protein
  • Gene expression
  • Pisum (seed protein)
  • Seed storage protein