Abstract
We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
- Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blochlinger K., Diggelmann H. Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Mol Cell Biol. 1984 Dec;4(12):2929–2931. doi: 10.1128/mcb.4.12.2929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bond-Matthews B., Davidson N. Transcription from each of the Drosophila act5C leader exons is driven by a separate functional promoter. Gene. 1988;62(2):289–300. doi: 10.1016/0378-1119(88)90566-5. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
- Chelsky D., Ralph R., Jonak G. Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol Cell Biol. 1989 Jun;9(6):2487–2492. doi: 10.1128/mcb.9.6.2487. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deuschle U., Pepperkok R., Wang F. B., Giordano T. J., McAllister W. T., Ansorge W., Bujard H. Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5400–5404. doi: 10.1073/pnas.86.14.5400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Friedman R. L., Stark G. R. alpha-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature. 1985 Apr 18;314(6012):637–639. doi: 10.1038/314637a0. [DOI] [PubMed] [Google Scholar]
- Fuerst T. R., Fernandez M. P., Moss B. Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2549–2553. doi: 10.1073/pnas.86.8.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hu M. C., Davidson N. The inducible lac operator-repressor system is functional for control of expression of injected DNA in Xenopus oocytes. Gene. 1988;62(2):301–313. doi: 10.1016/0378-1119(88)90567-7. [DOI] [PubMed] [Google Scholar]
- 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]
- Hynes N. E., Kennedy N., Rahmsdorf U., Groner B. Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2038–2042. doi: 10.1073/pnas.78.4.2038. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imbra R. J., Karin M. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C. Mol Cell Biol. 1987 Apr;7(4):1358–1363. doi: 10.1128/mcb.7.4.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karin M., Haslinger A., Heguy A., Dietlin T., Cooke T. Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity. Mol Cell Biol. 1987 Feb;7(2):606–613. doi: 10.1128/mcb.7.2.606. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karin M., Haslinger A., Holtgreve H., Cathala G., Slater E., Baxter J. D. Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA. Cell. 1984 Feb;36(2):371–379. doi: 10.1016/0092-8674(84)90230-7. [DOI] [PubMed] [Google Scholar]
- Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. M., Beato M. Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature. 1984 Apr 5;308(5959):513–519. doi: 10.1038/308513a0. [DOI] [PubMed] [Google Scholar]
- Labow M. A., Baim S. B., Shenk T., Levine A. J. Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol. 1990 Jul;10(7):3343–3356. doi: 10.1128/mcb.10.7.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee W., Haslinger A., Karin M., Tjian R. Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature. 1987 Jan 22;325(6102):368–372. doi: 10.1038/325368a0. [DOI] [PubMed] [Google Scholar]
- Lee W., Mitchell P., Tjian R. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell. 1987 Jun 19;49(6):741–752. doi: 10.1016/0092-8674(87)90612-x. [DOI] [PubMed] [Google Scholar]
- Liu H. S., Feliciano E. S., Stambrook P. J. Cytochemical observation of regulated bacterial beta-galactosidase gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9951–9955. doi: 10.1073/pnas.86.24.9951. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
- Moreau P., Hen R., Wasylyk B., Everett R., Gaub M. P., Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 1981 Nov 25;9(22):6047–6068. doi: 10.1093/nar/9.22.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Müeller-Storm H. P., Sogo J. M., Schaffner W. An enhancer stimulates transcription in trans when attached to the promoter via a protein bridge. Cell. 1989 Aug 25;58(4):767–777. doi: 10.1016/0092-8674(89)90110-4. [DOI] [PubMed] [Google Scholar]
- Müller M. M., Gerster T., Schaffner W. Enhancer sequences and the regulation of gene transcription. Eur J Biochem. 1988 Oct 1;176(3):485–495. doi: 10.1111/j.1432-1033.1988.tb14306.x. [DOI] [PubMed] [Google Scholar]
- Pace H. C., Lu P., Lewis M. lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1870–1873. doi: 10.1073/pnas.87.5.1870. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
- Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
- Sadler J. R., Sasmor H., Betz J. L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6785–6789. doi: 10.1073/pnas.80.22.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schüle R., Muller M., Kaltschmidt C., Renkawitz R. Many transcription factors interact synergistically with steroid receptors. Science. 1988 Dec 9;242(4884):1418–1420. doi: 10.1126/science.3201230. [DOI] [PubMed] [Google Scholar]
- Strähle U., Schmid W., Schütz G. Synergistic action of the glucocorticoid receptor with transcription factors. EMBO J. 1988 Nov;7(11):3389–3395. doi: 10.1002/j.1460-2075.1988.tb03212.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takahashi K., Vigneron M., Matthes H., Wildeman A., Zenke M., Chambon P. Requirement of stereospecific alignments for initiation from the simian virus 40 early promoter. Nature. 1986 Jan 9;319(6049):121–126. doi: 10.1038/319121a0. [DOI] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]