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. 1988 Sep 12;16(17):8467–8486. doi: 10.1093/nar/16.17.8467

Multiple copies of a G-rich element upstream of a cAMP-inducible Dictyostelium gene are necessary but not sufficient for efficient gene expression.

C J Pears 1, J G Williams 1
PMCID: PMC338570  PMID: 2843819

Abstract

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode co-ordinately expressed mRNA sequences which are inducible by extracellular cAMP. There are short, G-rich sequence elements upstream of both genes and we have previously shown that deletion of these elements from the CP2 gene abolishes cAMP-inducibility. We show here that the G-rich element from the CP1 gene is functionally homologous to that in the CP2 gene by reconstituting cAMP-inducibility in a deletion mutant of the CP2 gene using CP1-derived sequences. Both the CP1 and CP2 genes contain multiple G-rich elements. We show that efficient induction requires at least two copies of the CP1 element and that their relative orientation is unimportant. Two copies of an inverted relative orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.

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Selected References

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  1. Ayres K., Neuman W., Rowekamp W. G., Chung S. Developmental regulation of DNase I-hypersensitive sites in Dictyostelium discoideum. Mol Cell Biol. 1987 May;7(5):1823–1829. doi: 10.1128/mcb.7.5.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cohen S. M., Knecht D., Lodish H. F., Loomis W. F. DNA sequences required for expression of a Dictyostelium actin gene. EMBO J. 1986 Dec 1;5(12):3361–3366. doi: 10.1002/j.1460-2075.1986.tb04651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Datta S., Firtel R. A. Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum. Mol Cell Biol. 1987 Jan;7(1):149–159. doi: 10.1128/mcb.7.1.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Datta S., Gomer R. H., Firtel R. A. Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum. Mol Cell Biol. 1986 Mar;6(3):811–820. doi: 10.1128/mcb.6.3.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Driscoll D. M., Williams J. G. Two divergently transcribed genes of Dictyostelium discoideum are cyclic AMP-inducible and coregulated during development. Mol Cell Biol. 1987 Dec;7(12):4482–4489. doi: 10.1128/mcb.7.12.4482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dynan W. S., Tjian R. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell. 1983 Mar;32(3):669–680. doi: 10.1016/0092-8674(83)90053-3. [DOI] [PubMed] [Google Scholar]
  7. Early A. E., Williams J. G. Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum. Gene. 1987;59(1):99–106. doi: 10.1016/0378-1119(87)90270-8. [DOI] [PubMed] [Google Scholar]
  8. Everett R. D., Baty D., Chambon P. The repeated GC-rich motifs upstream from the TATA box are important elements of the SV40 early promoter. Nucleic Acids Res. 1983 Apr 25;11(8):2447–2464. doi: 10.1093/nar/11.8.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gidoni D., Kadonaga J. T., Barrera-Saldaña H., Takahashi K., Chambon P., Tjian R. Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. Science. 1985 Nov 1;230(4725):511–517. doi: 10.1126/science.2996137. [DOI] [PubMed] [Google Scholar]
  10. Jermyn K. A., Berks M., Kay R. R., Williams J. G. Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum. Development. 1987 Aug;100(4):745–755. doi: 10.1242/dev.100.4.745. [DOI] [PubMed] [Google Scholar]
  11. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  12. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Melton D. W., McEwan C., McKie A. B., Reid A. M. Expression of the mouse HPRT gene: deletional analysis of the promoter region of an X-chromosome linked housekeeping gene. Cell. 1986 Jan 31;44(2):319–328. doi: 10.1016/0092-8674(86)90766-x. [DOI] [PubMed] [Google Scholar]
  15. Nellen W., Firtel R. A. High-copy-number transformants and co-transformation in Dictyostelium. Gene. 1985;39(2-3):155–163. doi: 10.1016/0378-1119(85)90309-9. [DOI] [PubMed] [Google Scholar]
  16. Nellen W., Silan C., Firtel R. A. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Mol Cell Biol. 1984 Dec;4(12):2890–2898. doi: 10.1128/mcb.4.12.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nellen W., Silan C., Saur U., Firtel R. A. Regulatory sequences in the promoter of the Dictyostelium Actin 6 gene. EMBO J. 1986 Dec 1;5(12):3367–3372. doi: 10.1002/j.1460-2075.1986.tb04652.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Norris K., Norris F., Christiansen L., Fiil N. Efficient site-directed mutagenesis by simultaneous use of two primers. Nucleic Acids Res. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pears C. J., Mahbubani H. M., Williams J. G. Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discoideum. Nucleic Acids Res. 1985 Dec 20;13(24):8853–8866. doi: 10.1093/nar/13.24.8853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pears C. J., Williams J. G. Identification of a DNA sequence element required for efficient expression of a developmentally regulated and cAMP-inducible gene of Dictyostelium discoideum. EMBO J. 1987 Jan;6(1):195–200. doi: 10.1002/j.1460-2075.1987.tb04738.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Poole S. J., Firtel R. A. Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum. J Mol Biol. 1984 Jan 15;172(2):203–220. doi: 10.1016/s0022-2836(84)80038-8. [DOI] [PubMed] [Google Scholar]
  22. Romans P., Firtel R. A. Organization of the Dictyostelium discoideum actin multigene family. Flanking sequences show subfamily homologies and unusual dyad symmetries. J Mol Biol. 1985 Jun 5;183(3):311–326. doi: 10.1016/0022-2836(85)90003-8. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Struhl K. The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell. 1987 Sep 11;50(6):841–846. doi: 10.1016/0092-8674(87)90511-3. [DOI] [PubMed] [Google Scholar]
  25. Watts D. J., Ashworth J. M. Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J. 1970 Sep;119(2):171–174. doi: 10.1042/bj1190171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Williams J. G., Lloyd M. M. Changes in the abundance of polyadenylated RNA during slime mould development measured using cloned molecular hybridization probes. J Mol Biol. 1979 Mar 25;129(1):19–35. doi: 10.1016/0022-2836(79)90056-1. [DOI] [PubMed] [Google Scholar]
  27. Williams J. G., North M. J., Mahbubani H. A developmentally regulated cysteine proteinase in Dictyostelium discoideum. EMBO J. 1985 Apr;4(4):999–1006. doi: 10.1002/j.1460-2075.1985.tb03730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Williams J. G., Tsang A. S., Mahbubani H. A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7171–7175. doi: 10.1073/pnas.77.12.7171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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