Skip to main content
Log in

Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation

  • Research Article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Two genomic clones (λUbi-1 and λUbi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25°C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5′ untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5′ flanking region and the entire 5′ untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

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

Access this article

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. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley, New York (1988).

    Google Scholar 

  2. Ball E, Karlik CC, Beall CJ, Saville DL, Sparrow JC, Bullard B, Fyrberg EA: Arthrin, a myofibrillar protein of insect flight muscle is an actin-ubiquitin conjugate. Cell 51: 221–228 (1987).

    Article  PubMed  Google Scholar 

  3. Barsoum J, Varshavsky A: Preferential localization of variant nucleosomes near the 5′ end of the mouse dihydrofolate reductase gene. J Biol Chem 260: 7688–7697 (1985).

    PubMed  Google Scholar 

  4. Bond U, Schlesinger MJ: The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells. Mol Cell Biol 6: 4602–4610 (1986).

    PubMed  Google Scholar 

  5. Burke TJ, Callis J, Vierstra RD: Characterization of a polyubiquitin gene fromArabidopsis thaliana. Mol Gen Genet 213: 435–443 (1988).

    PubMed  Google Scholar 

  6. Callis J, Vierstra RD: Ubiquitin and ubiquitin genes in higher plants. Oxford Surv Plant Mol Cell Biol 6: 1–30 (1989).

    Google Scholar 

  7. Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells. Genes Devel 1: 1183–1200 (1987).

    PubMed  Google Scholar 

  8. Callis J, Fromm M, Walbot V: Heat inducible expression of a chimeric maize hsp70-CAT gene in maize protoplasts. Plant Physiol 88: 965–968 (1988).

    Google Scholar 

  9. Callis J, Raasch JA, Vierstra RD: Ubiquitin extension proteins ofArabidopsis thaliana. J Biol Chem 265: 12486–12493 (1990).

    PubMed  Google Scholar 

  10. Christensen AH, Quail PH: Sequence analysis and transcriptional regulation by heat shock of polyubiquitin transcripts from maize. Plant Mol Biol 12: 619–632 (1989).

    Google Scholar 

  11. Czarnecka E, Nagao RT, Key JL, Gurley WB: Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy metal induced inhibition of intron processing. Mol Cell Biol 8: 1113–1122 (1988).

    PubMed  Google Scholar 

  12. Dekeyser R, Claes B, Marichal M, VanMontagu M, Caplan A: Evaluation of selectable markers for rice transformation. Plant Physiol 90: 217–223 (1989).

    Google Scholar 

  13. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX: Nucl Acids Res 12: 387–395 (1984).

    PubMed  Google Scholar 

  14. Finley D, Bartel B, Varshavsky A: The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338: 394–401 (1989).

    Article  PubMed  Google Scholar 

  15. Finley D, Ozkaynak E, Varshavsky A: The yeast polyubiquitin gene is essential for resistance at high temperatures, starvation and other stresses. Cell 48: 1035–1046 (1987).

    Article  PubMed  Google Scholar 

  16. Fried VA, Smith HT, Hildebrandt E, Weiner K: Ubiquitin has instrinsic proteolytic activity: Implications for cellular regulation. Proc Natl Acad Sci USA 84: 3685–3689 (1987).

    PubMed  Google Scholar 

  17. Fromm M, Taylor LP, Walbot V: Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82: 5824–5828 (1985).

    PubMed  Google Scholar 

  18. Fromm M, Callis J, Taylor LP, Walbot V: Electroporation of DNA and RNA into plant protoplasts. Meth Enzymol 153: 351–366 (1987).

    Google Scholar 

  19. Gausing K, Barkardottir R: Structure and expression of ubiquitin genes in higher plants. Eur J Biochem 158: 57–62 (1986).

    PubMed  Google Scholar 

  20. Gilmour DS, Thomas GH, Elgin SCR:Drosophila nuclear proteins bind to regions of alternating C and T residues in gene promoters. Science 245: 1487–1490 (1989).

    PubMed  Google Scholar 

  21. Hanley BA, Schuler MA: Plant intron sequences: evidence for distinct groups of introns. Nucl Acids Res 16: 7159–7176 (1988).

    PubMed  Google Scholar 

  22. Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).

    Article  PubMed  Google Scholar 

  23. Hershko A: Ubiquitin-mediated protein degradation. J Biol Chem 263: 15237–15240 (1988).

    PubMed  Google Scholar 

  24. Jabben M, Shanklin J, Vierstra RD: Red light-induced accumulation of ubiquitin-phytochrome conjugated in both monocots and dicots. Plant Physiol 90: 380–384 (1989).

    Google Scholar 

  25. Jentsch S, McGrath JP, Varshavsky A: The yeast DNA repair gene RAD 6 encodes a ubiquitin-conjugating enzyme. Nature 329: 131–134 (1987).

    Article  PubMed  Google Scholar 

  26. Keith B, Chua N-H: Monocot and dicot pre-mRNAs are processed with different efficiencies in transgenic tobacco. EMBO J 5: 2149–2425 (1986).

    PubMed  Google Scholar 

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

    Google Scholar 

  28. Pelham HRB: A regulatory upstream promoter element inDrosophila HSP70 heat-shock gene. Cell 30: 517–528 (1982).

    Article  PubMed  Google Scholar 

  29. Rechsteiner M: Ubiquitin-mediated pathways for intracellular proteolysis. Annu Rev Cell Biol 3: 1–30 (1987).

    PubMed  Google Scholar 

  30. Rechsteiner M: Natural substrates of the ubiquitin proteolytic pathway. Cell 66: 615–618 (1991).

    Article  PubMed  Google Scholar 

  31. Redman KL, Rechsteiner M: Identification of the long ubiquitin extension as ribosomal protein S27a. Nature 338: 438–440 (1989).

    Article  PubMed  Google Scholar 

  32. Schlesinger M, Bond U: Ubiquitin genes. Oxford Surv Eukaryotic Genes 4: 77–89 (1987).

    Google Scholar 

  33. Shanklin J, Jabben M, Vierstra RD: Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation. Proc Natl Acad Sci USA 84: 359–363 (1987).

    Google Scholar 

  34. Sharp PM, Li W-H: Ubiquitin genes as a paradigm of concerted evolution of tandem repeats. J Mol Evol 25: 58–64 (1987).

    PubMed  Google Scholar 

  35. Sullivan TD, Christensen AH, Quail PH: Isolation and characterization of a maize chlorophyll a/b binding protein gene that produces high levels of mRNA in the dark. Mol Gen Genet 215: 431–440 (1989).

    PubMed  Google Scholar 

  36. Winter J, Wright R, Duck N, Gasser C, Fraley R, Shah D: The inhibition of petunia hsp70 mRNA processing during CdCl2 stress. Mol Gen Genet 211: 315–319 (1988).

    Article  Google Scholar 

  37. Yost HJ, Lindquist S: RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 45: 185–193 (1986).

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Christensen, A.H., Sharrock, R.A. & Quail, P.H. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18, 675–689 (1992). https://doi.org/10.1007/BF00020010

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020010

Key words

Navigation