1887

Abstract

SUMMARY

We constructed a recombinant DNA gene containing sequences from the 5′ flanking region of a dexamethasone-inducible rat growth hormone gene linked to the coding region of the polyoma virus transforming protein, the middle-size T (MT) antigen. We used this gene to derive cell lines in which the expression of the MT antigen could be regulated by dexamethasone. We transfected mouse NIH 3T3 cells and isolated transformed foci from cultures grown in the presence or absence of dexamethasone. The frequency of focus formation and the size of the transformed foci were increased in the presence of dexamethasone. Several transformants showed regulated expression of the MT antigen: the levels of polyoma-specific RNA, MT protein and MT-associated kinase activity were increased two- to fivefold in cells grown in the presence of dexamethasone. These results show that 248 base pairs of rat growth hormone DNA, including the first 237 base pairs upstream from the major transcription initiation site, contain promoter activity and a regulatory element required for glucocorticoid induction. This region of the rat growth hormone can be used to regulate expression of the polyoma MT antigen gene. In some cell lines regulated expression of the MT antigen was accompanied by regulated expression of transformed cell growth properties. The minimum level of the MT antigen required for expression of transformation was considerably less than the level found in a polyoma MT-transformed cell line. Increasing the level of the MT antigen led to increased expression of transformation, assayed by morphology, focus formation and growth in agar.

Keyword(s): expression , polyoma virus and T-antigen
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1985-10-01
2021-10-20
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References

  1. Barta A., Richards R. I., Baxter J. D., Shine I. 1981; Primary structure and evolution of rat growth hormone gene. Proceedings of the National Academy of Sciences, U.S.A 78:4867–4871
    [Google Scholar]
  2. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. 1984; Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell 38:767–777
    [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Green P. J., Betlach M. C., Heyneker H. L., Boyer H. W. 1977; Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
    [Google Scholar]
  4. Brinster R. L., Chen H. Y., Trumbauer M., Senear A. W., Warren R., Palmiter R. D. 1981; Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 21:223–231
    [Google Scholar]
  5. Brinster R. L., Chen H. Y., Warren R., Sarthy A., Palmiter R. D. 1982; Regulation of metallothionein-thymidine kinase fusion plasmids injected into mouse eggs. Nature, London 296:39–42
    [Google Scholar]
  6. Buetti E., Diggelmann H. 1981; Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones. Cell 23:335–345
    [Google Scholar]
  7. Buetti E., Diggelmann H. 1983; Glucocorticoid regulation of mouse mammary tumor virus: identification of a short essential DNA region. EMBO Journal 2:1423–1429
    [Google Scholar]
  8. Chandler V. L., Maler B. A., Yamamoto K. R. 1983; DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell 33:489–499
    [Google Scholar]
  9. Chirgwin J. M., Przybyla A. E., Macdonald R. J., Rutter W. J. 1979; Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299
    [Google Scholar]
  10. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. 1977; Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. Journal of Biological Chemistry 252:1102–1106
    [Google Scholar]
  11. Courtneidge S. A., Smith A. E. 1983; Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature, London 303:435–439
    [Google Scholar]
  12. Courtneidge S. A., Smith A. E. 1984; The complex of polyoma virus middle-T antigen and pp60c-src. EMBO Journal 3:585–591
    [Google Scholar]
  13. Dean D. C., Knoll B. J., Riser M. E., O’Malley B. W. 1983; A 5′-flanking sequence essential for progesterone regulation of an ovalbumin fusion gene. Nature, London 305:551–554
    [Google Scholar]
  14. Deininger P. L., Esty A., Laporte P., Hsu H., Friedmann T. 1980; The nucleotide sequence and restriction enzyme sites of the polyoma genome. Nucleic Acids Research 8:855–860
    [Google Scholar]
  15. Dobner P. R., Kawasaki E. S., Yu L. -Y., Bancroft F. C. 1981; Thyroid or glucocorticoid hormone induces pregrowth-hormone mRNA and its probable nuclear precursor in rat pituitary cells. Proceedings of the National Academy of Sciences, U. S. A 78:2230–2234
    [Google Scholar]
  16. Doehmer J., Barinaga M., Vale W., Rosenfeld M., Verma I. M., Evans R. M. 1982; Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation. Proceedings of the National Academy of Sciences, U.S.A 79:2268–2272
    [Google Scholar]
  17. Eckhart W., Hutchinson M. A., Hunter T. 1979; An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell 18:925–933
    [Google Scholar]
  18. Evans R. M., Birnberg N. C., Rosenfeld M. G. 1982; Glucocorticoid and thyroid hormones transcriptionally regulate growth hormone gene expression. Proceedings of the National Academy of Sciences, U.S.A 79:7659–7663
    [Google Scholar]
  19. Geisse S., Scheidereit C., Westphal H. M., Hynes N. E., Groner B., Beato M. 1982; Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumor virus DNA. EMBO Journal 1:1613–1619
    [Google Scholar]
  20. Heiser W., Eckhart W. 1982; Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells. Journal of Virology 44:175–188
    [Google Scholar]
  21. Hutchinson M. A., Hunter T., Eckhart W. 1978; Characterization of T-antigens in polyoma-infected and transformed cells. Cell 15:65–77
    [Google Scholar]
  22. Hynes N., Van Ooyen A. J. J., Kennedy N., Herrlich P., Ponta H., Groner B. 1983; Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene. Proceedings of the National Academy of Sciences, U.S.A 80:3637–3641
    [Google Scholar]
  23. Jakobovits E. B., Majors J. E., Varmus H. E. 1984; Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation. Cell 38:757–765
    [Google Scholar]
  24. Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. M., Beato M. 1984; Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature, London 308:513–519
    [Google Scholar]
  25. Klessig D. F., Brough D. E., Cleghon V. 1984; Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell. Molecular and Cellular Biology 4:1354–1362
    [Google Scholar]
  26. Lee F., Mulligan R., Berg P., Ringold G. 1981; Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature, London 294:228–232
    [Google Scholar]
  27. MacPherson I. 1964; Agar suspension culture for quantitation of transformed cells. In Fundamental Techniques in Virology pp 214–219 Edited by Habel K., Salzman N. P. New York & London: Academic Press;
    [Google Scholar]
  28. Majors J., Varmus H. E. 1983; A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. Proceedings of the National Academy of Sciences, U.S.A 80:5866–5870
    [Google Scholar]
  29. Maxam A. M., Gilbert W. 1980; Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65:499–560
    [Google Scholar]
  30. Meinkoth J., Wahl G. 1984; Hybridization of nucleic acids immobilized on solid supports. Analytical Biochemistry 138:267–284
    [Google Scholar]
  31. Miller A. D., Ong E. S., Rosenfeld M. G., Verma L. M., Evans R. M. 1984; Infectious and selectable retrovirus containing an inducible rat growth hormone minigene. Science 225:993–998
    [Google Scholar]
  32. Ostrowski M. C., Richard-Foy H., Wolford R. G., Berard D. S., Hager G. L. 1983; Glucocorticoid regulation of transcription at an amplified episomal promoter. Molecular and Cellular Biology 3:2045–2057
    [Google Scholar]
  33. Parks W. P., Ransom J. C., Young H. A., Scolnick E. M. 1975; Mammary tumor virus induction by glucocorticoids: characterization of specific transcriptional regulation. Journal of Biological Chemistry 250:3330–3336
    [Google Scholar]
  34. Payvar F., Defranco D., Firestone G. L., Edgar B., Wrange O., Okret S., Gustafsson J. A., Yamamoto K. R. 1983; Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35:381–392
    [Google Scholar]
  35. Pelham H. R. B. 1982; A regulatory upstream promoter element in the Drosophila Hsp 70 heat-shock gene. Cell 30:517–528
    [Google Scholar]
  36. Pfahl M. 1982; Specific binding of the glucocorticoid-receptor complex to the mouse mammary tumor proviral promoter region. Cell 31:475–482
    [Google Scholar]
  37. Pfahl M., Mcginnis D., Hendricks M., Groner B., Hynes N. E. 1983; Correlation of glucocorticoid receptor binding sites on MMTV proviral DNA with hormone inducible transcription. Science 222:1341–1343
    [Google Scholar]
  38. Ragg H., Weissmann C. 1983; Not more than 117 base pairs of 5′-flanking sequence are required for inducible expression of a human IFN-α gene. Nature, London 303:439–442
    [Google Scholar]
  39. Rassoulzadegan M., Cowie A., Carr A., Glaichenhaus N., Kamen R., Cuzin F. 1982; The roles of individual polyoma virus early proteins in oncogenic transformation. Nature, London 300:713–718
    [Google Scholar]
  40. Ringold G. M., Yamamoto K. R., Tomkins G. M., Bishop J. M., Varmus H. E. 1975; Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action. Cell 6:299–305
    [Google Scholar]
  41. Ringold G. M., Yamamoto K. R., Bishop J. M., Varmus H. E. 1977; Glucocorticoid-stimulated accumulation of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA. Proceedings of the National Academy of Sciences, U S A 74:2879–2883
    [Google Scholar]
  42. Robins D. M., Paek I., Axel R., Seeburg P. H. 1982a; Regulation of the human growth hormone gene in mouse cells. In Gene Regulation UCLA Symposia on Molecular and Cellular Biology, vol 26 pp 253–265 Edited by O’Malley B. W. New York & London: Academic Press;
    [Google Scholar]
  43. Robins D. M., Paek I., Seeburg P. H., Axel R. 1982b; Regulated expression of human growth hormone genes in mouse cells. Cell 29:623–631
    [Google Scholar]
  44. Schaffhausen B., Beniamin T. L. 1979; Phosphorylation of polyoma T antigens. Cell 18:935–946
    [Google Scholar]
  45. Schaffhausen B., Dorai K., Arakere G., Benjamin T. L. 1982; Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity. Molecular and Cellular Biology 2:1187–1198
    [Google Scholar]
  46. Scheidereit C., Geisse S., Westphal H. M., Beato M. 1983; The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature, London 304:749–752
    [Google Scholar]
  47. Schlegel R., Benjamin T. L. 1978; Cellular alterations dependent upon the polyoma virus hr-t function: separation of mitogenic from transforming capacities. Cell 14:587–599
    [Google Scholar]
  48. Smith A. E., Smith R., Griffin B., Fried M. 1979; Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell 18:915–924
    [Google Scholar]
  49. Sutcliffe J. G. 1978; pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Research 5:2721–2728
    [Google Scholar]
  50. Tavernier J., Gheysen D., Duerinck F., Vanderhayden J., Fiers W. 1983; Deletion mapping of the inducible promoter of human IFN-a gene. Nature, London 301:634–636
    [Google Scholar]
  51. Tisty T., Brown P. C., Johnston R., Schimke R. T. 1982; Enhanced frequency of generation of methoxtrexate resistance and gene amplification in cultured mouse and hamster cell lines. In Gene Amplification pp 231–238 Edited by Schimke R. T. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  52. Treisman R., Novak U., Favaloro J., Kamen R. 1981; Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature, London 292:595–600
    [Google Scholar]
  53. Tushinski R. J., Sussman P. M., Yu L. -Y., Bancroft F. C. 1977; Pregrowth hormone messenger RNA: glucocorticoid induction and identification in rat pituitary cells. Proceedings of the National Academy of Sciences, U.S.A 74:2357–2361
    [Google Scholar]
  54. Twigg A. J., Sherratt D. 1980; Trans-complementable copy-number mutants of plasmid ColEl. Nature, London 283:216–218
    [Google Scholar]
  55. Ucker D. F., Yamamoto K. R. 1984; Early events in the stimulation of MTV RNA synthesis by glucocorticoids: novel assays of transcription rates. Journal of Biological Chemistry 259:7416–7420
    [Google Scholar]
  56. Walter G., Hutchinson M. A., Hunter T., Eckhart W. 1982; Purification of polyoma virus medium-size tumor antigen by immunoaffinity chromatography. Proceedings of the National Academy of Sciences, U.S.A 79:4025–4029
    [Google Scholar]
  57. Weidle U., Weissmann C. 1983; The 5′-flanking region of a human IFN-a gene mediates viral induction of transcription. Nature, London 303:442–446
    [Google Scholar]
  58. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin C. 1979; DNA mediated transfer of the adenine phosphoribosyl-transferase locus into mammalian cells. Proceedings of the National Academy of Sciences, U.S.A 76:1373–1376
    [Google Scholar]
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