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. 1992 Sep;12(9):4038–4045. doi: 10.1128/mcb.12.9.4038

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

T Dingermann 1, H Werner 1, A Schütz 1, I Zündorf 1, K Nerke 1, D Knecht 1, R Marschalek 1
PMCID: PMC360295  PMID: 1508201

Abstract

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.

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Selected References

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  1. Allison D. S., Hall B. D. Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene. EMBO J. 1985 Oct;4(10):2657–2664. doi: 10.1002/j.1460-2075.1985.tb03984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold G. J., Gross H. J. Unrelated leader sequences can efficiently promote human tRNA gene transcription. Gene. 1987;51(2-3):237–246. doi: 10.1016/0378-1119(87)90312-x. [DOI] [PubMed] [Google Scholar]
  3. Arnold G. J., Schmutzler C., Gross H. J. Functional dissection of 5' and 3' extragenic control regions of human tRNA(Val) genes reveals two different regulatory effects. DNA. 1988 Mar;7(2):87–97. doi: 10.1089/dna.1988.7.87. [DOI] [PubMed] [Google Scholar]
  4. Bartholomew B., Kassavetis G. A., Geiduschek E. P. Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC. Mol Cell Biol. 1991 Oct;11(10):5181–5189. doi: 10.1128/mcb.11.10.5181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bertrand K. P., Postle K., Wray L. V., Jr, Reznikoff W. S. Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene. 1983 Aug;23(2):149–156. doi: 10.1016/0378-1119(83)90046-x. [DOI] [PubMed] [Google Scholar]
  6. Brent R., Ptashne M. A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene. Nature. 1984 Dec 13;312(5995):612–615. doi: 10.1038/312612a0. [DOI] [PubMed] [Google Scholar]
  7. Brink M., Gerisch G., Isenberg G., Noegel A. A., Segall J. E., Wallraff E., Schleicher M. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor. J Cell Biol. 1990 Oct;111(4):1477–1489. doi: 10.1083/jcb.111.4.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown M., Figge J., Hansen U., Wright C., Jeang K. T., Khoury G., Livingston D. M., Roberts T. M. lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells. Cell. 1987 Jun 5;49(5):603–612. doi: 10.1016/0092-8674(87)90536-8. [DOI] [PubMed] [Google Scholar]
  9. Choffat Y., Suter B., Behra R., Kubli E. Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes. Mol Cell Biol. 1988 Aug;8(8):3332–3337. doi: 10.1128/mcb.8.8.3332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  11. Dingermann T., Amon-Böhm E., Bertling W., Marschalek R., Nerke K. A family of non-allelic tRNA(ValGUU) genes from the cellular slime mold Dictyostelium discoideum. Gene. 1988 Dec 20;73(2):373–384. doi: 10.1016/0378-1119(88)90502-1. [DOI] [PubMed] [Google Scholar]
  12. Dingermann T., Brechner T., Marschalek R., Amon-Böhm E., Welker D. L. tRNAGlu(GAA) genes from the cellular slime mold Dictyostelium discoideum. DNA. 1989 Apr;8(3):193–204. doi: 10.1089/dna.1.1989.8.193. [DOI] [PubMed] [Google Scholar]
  13. Dingermann T., Burke D. J., Sharp S., Schaack J., Söll D. The 5- flanking sequences of Drosophila tRNAArg genes control their in vitro transcription in a Drosophila cell extract. J Biol Chem. 1982 Dec 25;257(24):14738–14744. [PubMed] [Google Scholar]
  14. Dingermann T., Frank-Stoll U., Werner H., Wissmann A., Hillen W., Jacquet M., Marschalek R. RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system. EMBO J. 1992 Apr;11(4):1487–1492. doi: 10.1002/j.1460-2075.1992.tb05193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dingermann T., Nerke K., Marschalek R. Influence of different 5'-flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae. Eur J Biochem. 1987 Dec 30;170(1-2):217–224. doi: 10.1111/j.1432-1033.1987.tb13689.x. [DOI] [PubMed] [Google Scholar]
  16. Dingermann T., Nerke K. Primer extension analysis of tRNA gene transcripts synthesized in vitro and in vivo. Anal Biochem. 1987 May 1;162(2):466–475. doi: 10.1016/0003-2697(87)90422-2. [DOI] [PubMed] [Google Scholar]
  17. Dingermann T., Reindl N., Brechner T., Werner H., Nerke K. Nonsense suppression in Dictyostelium discoideum. Dev Genet. 1990;11(5-6):410–417. doi: 10.1002/dvg.1020110514. [DOI] [PubMed] [Google Scholar]
  18. Dingermann T., Reindl N., Werner H., Hildebrandt M., Nellen W., Harwood A., Williams J., Nerke K. Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. Gene. 1989 Dec 28;85(2):353–362. doi: 10.1016/0378-1119(89)90428-9. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Figge J., Wright C., Collins C. J., Roberts T. M., Livingston D. M. Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells. Cell. 1988 Mar 11;52(5):713–722. doi: 10.1016/0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  21. Gatz C., Quail P. H. Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1394–1397. doi: 10.1073/pnas.85.5.1394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  23. Gouilloud E., Clarkson S. G. A dispersed tyrosine tRNA gene from Xenopus laevis with high transcriptional activity in vitro. J Biol Chem. 1986 Jan 5;261(1):486–494. [PubMed] [Google Scholar]
  24. Harloff C., Gerisch G., Noegel A. A. Selective elimination of the contact site A protein of Dictyostelium discoideum by gene disruption. Genes Dev. 1989 Dec;3(12A):2011–2019. doi: 10.1101/gad.3.12a.2011. [DOI] [PubMed] [Google Scholar]
  25. Hillen W., Schollmeier K., Gatz C. Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region. J Mol Biol. 1984 Jan 15;172(2):185–201. doi: 10.1016/s0022-2836(84)80037-6. [DOI] [PubMed] [Google Scholar]
  26. Hipskind R. A., Clarkson S. G. 5'-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene. Cell. 1983 Oct;34(3):881–890. doi: 10.1016/0092-8674(83)90545-7. [DOI] [PubMed] [Google Scholar]
  27. Hottinger H., Pearson D., Yamao F., Gamulin V., Cooley L., Cooper T., Söll D. Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae. Mol Gen Genet. 1982;188(2):219–224. doi: 10.1007/BF00332678. [DOI] [PubMed] [Google Scholar]
  28. Hu M. C., Davidson N. The inducible lac operator-repressor system is functional in mammalian cells. Cell. 1987 Feb 27;48(4):555–566. doi: 10.1016/0092-8674(87)90234-0. [DOI] [PubMed] [Google Scholar]
  29. Hudziak R. M., Laski F. A., RajBhandary U. L., Sharp P. A., Capecchi M. R. Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes. Cell. 1982 Nov;31(1):137–146. doi: 10.1016/0092-8674(82)90413-5. [DOI] [PubMed] [Google Scholar]
  30. Jung G., Hammer J. A., 3rd Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform. J Cell Biol. 1990 Jun;110(6):1955–1964. doi: 10.1083/jcb.110.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kassavetis G. A., Braun B. R., Nguyen L. H., Geiduschek E. P. S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. Cell. 1990 Jan 26;60(2):235–245. doi: 10.1016/0092-8674(90)90739-2. [DOI] [PubMed] [Google Scholar]
  32. Kassavetis G. A., Riggs D. L., Negri R., Nguyen L. H., Geiduschek E. P. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes. Mol Cell Biol. 1989 Jun;9(6):2551–2566. doi: 10.1128/mcb.9.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kuchino Y., Beier H., Akita N., Nishimura S. Natural UAG suppressor glutamine tRNA is elevated in mouse cells infected with Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1987 May;84(9):2668–2672. doi: 10.1073/pnas.84.9.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kumagai A., Hadwiger J. A., Pupillo M., Firtel R. A. Molecular genetic analysis of two G alpha protein subunits in Dictyostelium. J Biol Chem. 1991 Jan 15;266(2):1220–1228. [PubMed] [Google Scholar]
  35. Laski F. A., Belagaje R., Hudziak R. M., Capecchi M. R., Norton G. P., Palese P., RajBhandary U. L., Sharp P. A. Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells. EMBO J. 1984 Nov;3(11):2445–2452. doi: 10.1002/j.1460-2075.1984.tb02154.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lofquist A., Sharp S. The 5'-flanking sequences of Drosophila melanogaster tRNA5Asn genes differentially arrest RNA polymerase III. J Biol Chem. 1986 Nov 5;261(31):14600–14606. [PubMed] [Google Scholar]
  37. Marschalek R., Dingermann T. Identification of a protein factor binding to the 5'-flanking region of a tRNA gene and being involved in modulation of tRNA gene transcription in vivo in Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Jul 25;16(14B):6737–6752. doi: 10.1093/nar/16.14.6737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Raymond K. C., Raymond G. J., Johnson J. D. In vivo modulation of yeast tRNA gene expression by 5'-flanking sequences. EMBO J. 1985 Oct;4(10):2649–2656. doi: 10.1002/j.1460-2075.1985.tb03983.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Romans P., Firtel R. A., Saxe C. L., 3rd Gene-specific expression of the actin multigene family of Dictyostelium discoideum. J Mol Biol. 1985 Nov 20;186(2):337–355. doi: 10.1016/0022-2836(85)90109-3. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Sharp P. M., Devine K. M. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons. Nucleic Acids Res. 1989 Jul 11;17(13):5029–5039. doi: 10.1093/nar/17.13.5029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sharp S. J., Schaack J., Cooley L., Burke D. J., Söll D. Structure and transcription of eukaryotic tRNA genes. CRC Crit Rev Biochem. 1985;19(2):107–144. doi: 10.3109/10409238509082541. [DOI] [PubMed] [Google Scholar]
  44. Shaw K. J., Olson M. V. Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene. Mol Cell Biol. 1984 Apr;4(4):657–665. doi: 10.1128/mcb.4.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sprague K. U., Hagenbüchle O., Zuniga M. C. The nucleotide sequence of two silk gland alanine tRNAs: implications for fibroin synthesis and for initiator tRNA structure. Cell. 1977 Jul;11(3):561–570. doi: 10.1016/0092-8674(77)90074-5. [DOI] [PubMed] [Google Scholar]
  46. Sprague K. U., Larson D., Morton D. 5' flanking sequence signals are required for activity of silkworm alanine tRNA genes in homologous in vitro transcription systems. Cell. 1980 Nov;22(1 Pt 1):171–178. doi: 10.1016/0092-8674(80)90165-8. [DOI] [PubMed] [Google Scholar]
  47. Stutz F., Gouilloud E., Clarkson S. G. Oocyte and somatic tyrosine tRNA genes in Xenopus laevis. Genes Dev. 1989 Aug;3(8):1190–1198. doi: 10.1101/gad.3.8.1190. [DOI] [PubMed] [Google Scholar]
  48. Sun T. J., Devreotes P. N. Gene targeting of the aggregation stage cAMP receptor cAR1 in Dictyostelium. Genes Dev. 1991 Apr;5(4):572–582. doi: 10.1101/gad.5.4.572. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Willis I., Nichols M., Chisholm V., Söll D., Heyer W. D., Szankasi P., Amstutz H., Munz P., Kohli J. Functional complementation between mutations in a yeast suppressor tRNA gene reveals potential for evolution of tRNA sequences. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7860–7864. doi: 10.1073/pnas.83.20.7860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Witke W., Nellen W., Noegel A. Homologous recombination in the Dictyostelium alpha-actinin gene leads to an altered mRNA and lack of the protein. EMBO J. 1987 Dec 20;6(13):4143–4148. doi: 10.1002/j.1460-2075.1987.tb02760.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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