Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Jan;3(1):235–243. doi: 10.1002/j.1460-2075.1984.tb01790.x

cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells

C Savakis 1,1, M M D Koehler 1, P Cherbas 1,*
PMCID: PMC557326  PMID: 16453496

Abstract

The ecdysone-inducible polypeptides (EIPs) 28, 29 and 40 were identified previously as polypeptides whose synthesis is stimulated early in the ecdysone response of Drosophila Kc cells. We have now shown, using two-dimensional gels, that each of these EIPs consists of three species differing in pI, and all stimulated by ecdysone. Translations and hybrid-arrested translations indicated that the poly(A)+ EIP mRNAs increase ˜10-fold in abundance during the first 4 h of ecdysone treatment. By a differential screen of a cDNA library we have identified cDNA clones corresponding to all three EIPs. Two kinds of clones were isolated: one hybridizes to the EIP 40 mRNA(s); the second hybridizes to the mRNA(s) encoding all the EIPs 28 and 29. The EIP 28/29 and EIP 40 loci detected by these clones are each present at single sites on the polytene chromosomes and each is at or in the vicinity of an ecdysone-regulated puff.

Keywords: ecdysone, Drosophila, cDNA clones, Kc cells, steroid

Full text

PDF
235

Images in this article

236Fig. 1.
on p.236
237Fig. 2.
on p.237
238Fig. 3.
on p.238
238Fig. 4.
on p.238
239Fig. 5.
on p.239

Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashburner M. Patterns of puffing activity in the salivary gland chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of D. melanogaster cultured in vitro. Chromosoma. 1972;38(3):255–281. doi: 10.1007/BF00290925. [DOI] [PubMed] [Google Scholar]
  3. Ashburner M. Puffing patterns in Drosophila melanogaster and related species. Results Probl Cell Differ. 1972;4:101–151. doi: 10.1007/978-3-540-37164-9_5. [DOI] [PubMed] [Google Scholar]
  4. Ashburner M. Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration. Dev Biol. 1973 Nov;35(1):47–61. doi: 10.1016/0012-1606(73)90006-7. [DOI] [PubMed] [Google Scholar]
  5. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  6. Berger E., Cox G. A precursor of cytoplasmic actin in cultured Drosophila cells. J Cell Biol. 1979 Jun;81(3):680–683. doi: 10.1083/jcb.81.3.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berger E., Ringler R., Alahiotis S., Frank M. Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures. Dev Biol. 1978 Feb;62(2):498–511. doi: 10.1016/0012-1606(78)90231-2. [DOI] [PubMed] [Google Scholar]
  8. Best-Belpomme M., Courgeon A. M. Ecdysterone and acetylcholinesterase activity in cultured Drosophila cells. Inducible, non-inducible and constitutive clones or lines. FEBS Lett. 1977 Oct 15;82(2):345–347. doi: 10.1016/0014-5793(77)80617-0. [DOI] [PubMed] [Google Scholar]
  9. Best-Belpomme M., Courgeon A. M., Rambach A. beta-Galactosidase is induced by hormone in Drosophila melanogaster cell cultures. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6102–6106. doi: 10.1073/pnas.75.12.6102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  11. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  12. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  13. Buetti E., Diggelmann H. Glucocorticoid regulation of mouse mammary tumor virus: identification of a short essential DNA region. EMBO J. 1983;2(8):1423–1429. doi: 10.1002/j.1460-2075.1983.tb01601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chandler V. L., Maler B. A., Yamamoto K. R. DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell. 1983 Jun;33(2):489–499. doi: 10.1016/0092-8674(83)90430-0. [DOI] [PubMed] [Google Scholar]
  15. Cherbas P., Cherbas L., Williams C. M. Induction of acetylcholinesterase activity by beta-ecdysone in a Drosophila cell line. Science. 1977 Jul 15;197(4300):275–277. doi: 10.1126/science.877552. [DOI] [PubMed] [Google Scholar]
  16. Courgeon A. M. Action of insect hormones at the cellular level. Morphological changes of a diploid cell line of Drosophila melanogaster, treated with ecdysone and several analogues in vitro. Exp Cell Res. 1972 Oct;74(2):327–336. doi: 10.1016/0014-4827(72)90384-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Echalier G., Ohanessian A. In vitro culture of Drosophila melanogaster embryonic cells. In Vitro. 1970 Nov-Dec;6(3):162–172. doi: 10.1007/BF02617759. [DOI] [PubMed] [Google Scholar]
  19. Fasel N., Pearson K., Buetti E., Diggelmann H. The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription. EMBO J. 1982;1(1):3–7. doi: 10.1002/j.1460-2075.1982.tb01115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geisse S., Scheidereit C., Westphal H. M., Hynes N. E., Groner B., Beato M. Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumour virus DNA. EMBO J. 1982;1(12):1613–1619. doi: 10.1002/j.1460-2075.1982.tb01363.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Govindan M. V., Spiess E., Majors J. Purified glucocorticoid receptor-hormone complex from rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in vitro. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5157–5161. doi: 10.1073/pnas.79.17.5157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hanahan D., Meselson M. Plasmid screening at high colony density. Gene. 1980 Jun;10(1):63–67. doi: 10.1016/0378-1119(80)90144-4. [DOI] [PubMed] [Google Scholar]
  24. Hastie N. D., Held W. A. Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1217–1221. doi: 10.1073/pnas.75.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Higgins S. J., Gehring U. Molecular mechanisms of steroid hormone action. Adv Cancer Res. 1978;28:313–397. doi: 10.1016/s0065-230x(08)60650-8. [DOI] [PubMed] [Google Scholar]
  26. Holmes D. S., Bonner J. Preparation, molecular weight, base composition, and secondary structure of giant nuclear ribonucleic acid. Biochemistry. 1973 Jun 5;12(12):2330–2338. doi: 10.1021/bi00736a023. [DOI] [PubMed] [Google Scholar]
  27. Huang A. L., Ostrowski M. C., Berard D., Hager G. L. Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981 Dec;27(2 Pt 1):245–255. doi: 10.1016/0092-8674(81)90408-6. [DOI] [PubMed] [Google Scholar]
  28. Hynes N., van Ooyen A. J., Kennedy N., Herrlich P., Ponta H., Groner B. Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3637–3641. doi: 10.1073/pnas.80.12.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ireland R. C., Berger E. M. Synthesis of low molecular weight heat shock peptides stimulated by ecdysterone in a cultured Drosophila cell line. Proc Natl Acad Sci U S A. 1982 Feb;79(3):855–859. doi: 10.1073/pnas.79.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  32. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  33. Majors J., Varmus H. E. A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5866–5870. doi: 10.1073/pnas.80.19.5866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McKeehan W. L. Regulation of hemoglobin synthesis. Effect of concentration of messenger ribonucleic acid, ribosome subunits, initiation factors, and salts on ratio of alpha and beta chains synthesized in vitro. J Biol Chem. 1974 Oct 25;249(20):6517–6526. [PubMed] [Google Scholar]
  35. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  36. Palmiter R. D. Differential rates of initiation of conalbumin and ovalbumin messenger ribonucleic acid in reticulocyte lysates. J Biol Chem. 1974 Nov 10;249(21):6779–6787. [PubMed] [Google Scholar]
  37. Parker M. Enhancer elements activated by steroid hormones? Nature. 1983 Aug 25;304(5928):687–688. doi: 10.1038/304687a0. [DOI] [PubMed] [Google Scholar]
  38. Paterson B. M., Bishop J. O. Changes in the mRNA population of chick myoblasts during myogenesis in vitro. Cell. 1977 Nov;12(3):751–765. doi: 10.1016/0092-8674(77)90275-6. [DOI] [PubMed] [Google Scholar]
  39. Payvar F., Wrange O., Carlstedt-Duke J., Okret S., Gustafsson J. A., Yamamoto K. R. Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6628–6632. doi: 10.1073/pnas.78.11.6628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  41. Ploegh H. L., Orr H. T., Strominger J. L. Molecular cloning of a human histocompatibility antigen cDNA fragment. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6081–6085. doi: 10.1073/pnas.77.10.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ponta H., Kennedy N., Herrlich P., Hynes N. E., Groner B. A deletion mutant of mouse mammary tumour virus, lacking 516 nucleotides of the 5' long terminal repeat sequence, can be expressed in a hormone-responsive fashion. J Gen Virol. 1983 Mar;64(Pt 3):567–577. doi: 10.1099/0022-1317-64-3-567. [DOI] [PubMed] [Google Scholar]
  43. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Robb J. A. Maintenance of imaginal discs of Drosophila melanogaster in chemically defined media. J Cell Biol. 1969 Jun;41(3):876–885. doi: 10.1083/jcb.41.3.876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rosset R. Effects of ecdysone on a Drosophila cell line. Exp Cell Res. 1978 Jan;111(1):31–36. doi: 10.1016/0014-4827(78)90233-1. [DOI] [PubMed] [Google Scholar]
  47. Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Apr;3(4):863–877. doi: 10.1093/nar/3.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Savakis C., Demetri G., Cherbas P. Ecdysteroid-inducible polypeptides in a Drosophila cell line. Cell. 1980 Dec;22(3):665–674. doi: 10.1016/0092-8674(80)90542-5. [DOI] [PubMed] [Google Scholar]
  49. Scheidereit C., Geisse S., Westphal H. M., Beato M. The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature. 1983 Aug 25;304(5928):749–752. doi: 10.1038/304749a0. [DOI] [PubMed] [Google Scholar]
  50. Stewart A. G., Gander E. S., Morel C., Luppis B., Scherrer K. Differential translation of duck- and rabbit-globin messenger RNAs in reticulocyte-lysate systems. Eur J Biochem. 1973 Apr;34(2):205–212. doi: 10.1111/j.1432-1033.1973.tb21105.x. [DOI] [PubMed] [Google Scholar]
  51. Ucker D. S., Ross S. R., Yamamoto K. R. Mammary tumor virus DNA contains sequences required for its hormone-regulated transcription. Cell. 1981 Dec;27(2 Pt 1):257–266. doi: 10.1016/0092-8674(81)90409-8. [DOI] [PubMed] [Google Scholar]
  52. Velissariou V., Ashburner M. Cytogenetic and genetic mapping of a salivary gland secretion protein in Drosophila melanogaster. Chromosoma. 1981;84(2):173–185. doi: 10.1007/BF00399129. [DOI] [PubMed] [Google Scholar]
  53. Vournakis J. N., Efstratiadis A., Kafatos F. C. Electrophoretic patterns of deadenylylated chorion and globin mRNAs. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2959–2963. doi: 10.1073/pnas.72.8.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wensink P. C., Finnegan D. J., Donelson J. E., Hogness D. S. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. doi: 10.1016/0092-8674(74)90045-2. [DOI] [PubMed] [Google Scholar]
  55. Wyss C. Juvenile hormone analogue counteracts growth stimulation and inhibition by ecdysones in clonal Drosophila cell line. Experientia. 1976 Oct 15;32(10):1272–1274. doi: 10.1007/BF01953088. [DOI] [PubMed] [Google Scholar]
  56. Yamamoto K. R., Alberts B. M. Steroid receptors: elements for modulation of eukaryotic transcription. Annu Rev Biochem. 1976;45:721–746. doi: 10.1146/annurev.bi.45.070176.003445. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES