Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Feb 11;16(3):1043–1061. doi: 10.1093/nar/16.3.1043

Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells.

T A Bunch 1, Y Grinblat 1, L S Goldstein 1
PMCID: PMC334736  PMID: 3125519

Abstract

The promoter from the metallothionein gene may be a useful conditional promoter for the construction of chimeric genes to be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed chimeric gene containing the metallothionein promoter were examined. Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels. The level of induction depends on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing the metallothionein promoter shows a similar induction when transformed into the cells.

Full text

PDF
1043

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen R. D., Weser U. Partial purification, characterization and translation in vitro of rat liver metallothionein messenger ribonucleic acid. Biochem J. 1978 Dec 1;175(3):841–852. doi: 10.1042/bj1750841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  3. Bourouis M., Jarry B. Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance. EMBO J. 1983;2(7):1099–1104. doi: 10.1002/j.1460-2075.1983.tb01552.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brinster R. L., Chen H. Y., Warren R., Sarthy A., Palmiter R. D. Regulation of metallothionein--thymidine kinase fusion plasmids injected into mouse eggs. Nature. 1982 Mar 4;296(5852):39–42. doi: 10.1038/296039a0. [DOI] [PubMed] [Google Scholar]
  5. Carter A. D., Felber B. K., Walling M. J., Jubier M. F., Schmidt C. J., Hamer D. H. Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7392–7396. doi: 10.1073/pnas.81.23.7392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cherian M. G., Goyer R. A. Methallothioneins and their role in the metabolism and toxicity of metals. Life Sci. 1978 Jul 3;23(1):1–9. doi: 10.1016/0024-3205(78)90317-x. [DOI] [PubMed] [Google Scholar]
  7. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Debec A., Mokdad R., Wegnez M. Metallothioneins and resistance to cadmium poisoning in Drosophila cells. Biochem Biophys Res Commun. 1985 Feb 28;127(1):143–152. doi: 10.1016/s0006-291x(85)80137-6. [DOI] [PubMed] [Google Scholar]
  9. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  10. Durnam D. M., Palmiter R. D. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem. 1981 Jun 10;256(11):5712–5716. [PubMed] [Google Scholar]
  11. Enger M. D., Rall L. B., Hildebrand C. E. Thionein gene expression in Cd++-variants of the CHO cell: correlation of thionein synthesis rates with translatable mRNA levels during induction, deinduction, and superinduction. Nucleic Acids Res. 1979 Sep 11;7(1):271–288. doi: 10.1093/nar/7.1.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldberg D. A. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5794–5798. doi: 10.1073/pnas.77.10.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goldstein L. S., Laymon R. A., McIntosh J. R. A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences. J Cell Biol. 1986 Jun;102(6):2076–2087. doi: 10.1083/jcb.102.6.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamer D. H., Walling M. Regulation in vivo of a cloned mammalian gene: cadmium induces the transcription of a mouse metallothionein gene in SV40 vectors. J Mol Appl Genet. 1982;1(4):273–288. [PubMed] [Google Scholar]
  15. Karin M., Andersen R. D., Herschman H. R. Induction of metallothionein mRNA in HeLa cells by dexamethasone and by heavy metals. Eur J Biochem. 1981 Sep 1;118(3):527–531. doi: 10.1111/j.1432-1033.1981.tb05551.x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kreitman M. Nucleotide polymorphism at the alcohol dehydrogenase locus of Drosophila melanogaster. Nature. 1983 Aug 4;304(5925):412–417. doi: 10.1038/304412a0. [DOI] [PubMed] [Google Scholar]
  18. Kwan S. P., Wood T. G., Lingrel J. B. Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells. Proc Natl Acad Sci U S A. 1977 Jan;74(1):178–182. doi: 10.1073/pnas.74.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levinson W., Oppermann H., Jackson J. Transition series metals and sulfhydryl reagents induce the synthesis of four proteins in eukaryotic cells. Biochim Biophys Acta. 1980;606(1):170–180. doi: 10.1016/0005-2787(80)90108-2. [DOI] [PubMed] [Google Scholar]
  20. Maroni G., Otto E., Lastowski-Perry D. Molecular and cytogenetic characterization of a metallothionein gene of Drosophila. Genetics. 1986 Mar;112(3):493–504. doi: 10.1093/genetics/112.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mayo K. E., Warren R., Palmiter R. D. The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell. 1982 May;29(1):99–108. doi: 10.1016/0092-8674(82)90094-0. [DOI] [PubMed] [Google Scholar]
  22. Ohi S., Cardenosa G., Pine R., Huang P. C. Cadmium-induced accumulation of metallothionein messenger RNA in rat liver. J Biol Chem. 1981 Mar 10;256(5):2180–2184. [PubMed] [Google Scholar]
  23. Olafson R. W., Abel K., Sim R. G. Prokaryotic metallothionein: preliminary characterization of a blue-green alga heavy metal-binding protein. Biochem Biophys Res Commun. 1979 Jul 12;89(1):36–43. doi: 10.1016/0006-291x(79)90939-2. [DOI] [PubMed] [Google Scholar]
  24. Otto E., Allen J. M., Young J. E., Palmiter R. D., Maroni G. A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn. Mol Cell Biol. 1987 May;7(5):1710–1715. doi: 10.1128/mcb.7.5.1710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Richards M. P., Cousins R. J. Isolation of an intestinal metallothionein induced by parenteral zinc. Biochem Biophys Res Commun. 1977 Mar 21;75(2):286–294. doi: 10.1016/0006-291x(77)91041-5. [DOI] [PubMed] [Google Scholar]
  26. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  27. Savakis C., Koehler M. M., Cherbas P. cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells. EMBO J. 1984 Jan;3(1):235–243. doi: 10.1002/j.1460-2075.1984.tb01790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  29. Shapiro S. G., Squibb K. S., Markowitz L. A., Cousins R. J. Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid. Biochem J. 1978 Dec 1;175(3):833–840. doi: 10.1042/bj1750833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Squibb K. S., Cousins R. J. Synthesis of metallothionein in a polysomal cell-free system. Biochem Biophys Res Commun. 1977 Apr 11;75(3):806–812. doi: 10.1016/0006-291x(77)91544-3. [DOI] [PubMed] [Google Scholar]
  31. Stuart G. W., Searle P. F., Chen H. Y., Brinster R. L., Palmiter R. D. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. doi: 10.1073/pnas.81.23.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vaslet C. A., O'Connell P., Izquierdo M., Rosbash M. Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster. Nature. 1980 Jun 26;285(5767):674–676. doi: 10.1038/285674a0. [DOI] [PubMed] [Google Scholar]
  34. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES