Characterization of the Human N-ras Promoter Region

  • J. T. Thorn
  • A. V. Todd
  • D. Warrilow
  • F. Watt
  • P. L. Molloy
  • H. J. Iland
Part of the NATO ASI Series book series (NSSA, volume 220)


Overexpression of ras protooncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer. A significant proportion of promoter activity was found to lie within a 438bp fragment comprising an untranslated exon (exon -1) with adjacent 5′ sequence and a small CpG island. A 107bp fragment at the 5′ end of exon -1 was essential for promoter activity, while a 44bp deletion from within this region decreased promoter activity by two thirds. Unlike the human H-ras promoter, the human N-ras promoter did not exhibit bidirectional activity. DNase footprinting of the 438bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb, and E4TF1). Using purified MLTF and appropriate competitors in gel shift and DNase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon -1.


K562 Cell Negative Regulatory Element Untranslated Exon Major Late Promoter Human Fibrosarcoma Cell Line HT1080 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Barbacid, M. 1987, Ras genes, Ann. Rev. Biochem., 56:779.PubMedCrossRefGoogle Scholar
  2. 2.
    Biedenkapp, H., Borgmeyer, U., Sippel, A. E., & Klempnauer, K.-H. 1988, Viral myb oncogene encodes a sequence-specific DNA-binding activity, Nature, 335:835.PubMedCrossRefGoogle Scholar
  3. 3.
    Bird, A. P. 1986, CpG-rich islands and the function of DNA methylation, Nature, 321:209.PubMedCrossRefGoogle Scholar
  4. 4.
    Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., & Weintraub, H. 1990, Sequence-specific DNA binding by the c-myc protein, Science, 250:1149.PubMedCrossRefGoogle Scholar
  5. 5.
    Brown, R., Marshall, C. J., Pennie, S. G., & Hall, A. 1984, Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080, EMBO J, 3:1321.PubMedGoogle Scholar
  6. 6.
    Carthew, R. W., Chodosh, L. A., & Sharp, P. A. 1985, An RNA polymerase II transcription factor binds to an upstream element in the Adenovirus major late promoter, Cell, 43:439.PubMedCrossRefGoogle Scholar
  7. 7.
    Cohen, J. B., & Levison, A. D. 1988, A point mutation in the last intron responsible for increased expression and transforming activity of the c-H-ras oncogene, Nature, 334:119.PubMedCrossRefGoogle Scholar
  8. 8.
    Croaker, G. M., Wass, E. J., & Hand, H. J. 1990, Electric field-mediated gene transfer into K562 cells: optimization of parameters affecting efficiency, Leukemia, 4:502.PubMedGoogle Scholar
  9. 9.
    Doniger, J. 1987, Differential conservation of non-coding regions within human and guinea pig N-ras genes, Oncogene, 1:331.PubMedGoogle Scholar
  10. 10.
    Doniger, J., & DiPaolo, J. A. 1988, Coordinate N-ras mRNA up-regu;ation with mutational activation in tumorigenic guinea pig cells, Nucleic Acids Res., 16:969.PubMedCrossRefGoogle Scholar
  11. 11.
    Gardiner-Garden, M., & Frommer, M. 1987, CpG islands in vertebrate genomes, J. Mol. Biol., 196:261.PubMedCrossRefGoogle Scholar
  12. 12.
    Gorman, C., Padmanabhan, R., & Howard, B. H. 1983, High efficiency DNA-mediated transformation of primate cells, Nature, 221:551.Google Scholar
  13. 13.
    Gerosa, M. A., Talarico, D., Fognani, C., Raimondi, E., Colombatti, M., Tridente, G., De Carli, L., & Della Valle, G. 1989, Overexpression of N-ras oncogene and epidermal growth factor receptor gene in human glioblastomas, J Natl. Cancer Inst., 81:63.PubMedCrossRefGoogle Scholar
  14. 14.
    Hai, T., Liu, F., Coukos, W. J., & Green, M. R. 1989, Transcriptional factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers, Genes Dev., 3:2083.PubMedCrossRefGoogle Scholar
  15. 15.
    Halazonetis, T. D., Georgopoulos, K., Greenberg, M. E., & Leder, P. 1988, c-jun dimerizes with itself and with c-fos, forming complexes of different DNA binding affinities, Cell, 55:917.PubMedCrossRefGoogle Scholar
  16. 16.
    Hall, A., & Brown, R. 1985, Human N-ras: cDNA cloning and gene structure, Nucleic Acids Res., 13:5255.PubMedCrossRefGoogle Scholar
  17. 7.
    Hofler, H., Ruhri, C., Putz, B., Wirnsberger, G., & Hauser, H. 1988, Oncogene expression in endocrine pancreatic tumors, Virchows. Archiv. B Cell Pathology, 55:355.Google Scholar
  18. 18.
    Jones, N. C., Rigby, P. W. J., & Ziff, E. B. 1988, Transacting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses, Genes Dev., 2:267.PubMedCrossRefGoogle Scholar
  19. 19.
    Lozzio, C. B., & Lozzio, B. B. 1975, Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome, Blood, 45:321.PubMedGoogle Scholar
  20. 20.
    Maxam, A. M., & Gilbert, W. 1980, Sequencing end-labelled DNA with base-specific chemical cleavage, Methods Enzymol., 65:499.PubMedCrossRefGoogle Scholar
  21. 21.
    McKay, I. A., Marshall, C. J., Cales, C. & Hall, A. 1986, Transformation and stimulation of DNA synthesis in NIH-3T3 cells are a titratable function of normal p21N-ras expression, EMBO J, 5:2617.PubMedGoogle Scholar
  22. 22.
    Mitchell, P. J., & Tjian, R. 1989, Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins, Science, 245:371.PubMedCrossRefGoogle Scholar
  23. 23.
    Miyamoto, N. G., Moncollin, V., Egly, J. M., & Chambon, P. 1985, Specific interaction between a transcription factor and the upstream element of the Adenovirus-2 major late promoter, EMBO J, 4:3563.PubMedGoogle Scholar
  24. 24.
    Paciucci, R., & Pellicer, A. 1991, Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element, Mol. Cell. Biol., 11:1334.PubMedGoogle Scholar
  25. 25.
    Prendergast, G. C., & Ziff, E. B. 1991, Methylation-sensitive sequence-specific DNA binding by the c-myc basic region, Science, 251:186.PubMedCrossRefGoogle Scholar
  26. 26.
    Sawadogo, M., & Roeder, R. G. 1986, DNA-binding specificity of USF, a human gene-specific transcription factor requirred for maximal expression of the major late promoter of Adenovirus, In Cancer cells: DNA tumor viruses 4:147, Cold Spring Laboratories, Cold Spring Habor New York.Google Scholar
  27. 27.
    Sharpiro, D. J., Sharp, P. A., Wahli, W. W., & Keller, M. J. 1988, A high-efficiency HeLa cell nuclear transcription extract, DNA, 7:47.CrossRefGoogle Scholar
  28. 28.
    Sleigh, M. J. 1986, A nonchromatogrphic assay for expression of the chloramphenicol acetyltransferase gene in eukaryotic cells, Anal. Biochem., 156:251.PubMedCrossRefGoogle Scholar
  29. 29.
    Suarez, H. G., Daya-Grosjean, L., Schlaifer, D., Nardeux, P., Renault, G., Bos, J. L., & Sarasin, A. 1989, Activated oncogenes in human skin tumors from a repair-deficient syndrome, xeroderma pigmentosum, Cancer Res., 49:1223.PubMedGoogle Scholar
  30. 30.
    Tanaka, T., Slamon, D. J., Battifora, H., & Cline, M. J. 1986, Expression of p21 ras oncoproteins in human cancers, Cancer Res., 46:1465.PubMedGoogle Scholar
  31. 31.
    Taparowski, E., Shimizu, K., Goldfarb, M., & Wigier, M. 1983, Structure and activation of the human N-ras gene, Cell, 34:581.CrossRefGoogle Scholar
  32. 32.
    Thomson, T. M., Green, S. H., Trotta, R. J., Burstein, D. E., & Pellicer, A. 1990, Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line, Mol. Cell. Biol., 10:1556.PubMedGoogle Scholar
  33. 33.
    Thor, A., Horan Hand, P., Wunderlich, D., Caruso, A., Muraro, R., & Schlom, J. 1984, Monoclonal antibodies define differential ras expression in malignant and benign colonic diseases, Nature, 311:562.PubMedCrossRefGoogle Scholar
  34. 34.
    Thorn, J. T., Todd, A. V., Warrilow, D., Watt, F., Molloy, P. L., & Iland, H. J. 1991, Characterization of the human N-ras promoter region, Oncogene, in press.Google Scholar
  35. 35.
    Watt, F., & Molloy, P. L. 1988, Cytosine methylation prevents binding to DNA of a HeLa cell transcription afctor requirred for optimal expression of the Adenovirus major late promoter, Genes Dev., 2:1136.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • J. T. Thorn
    • 1
  • A. V. Todd
    • 1
  • D. Warrilow
    • 1
  • F. Watt
    • 2
  • P. L. Molloy
    • 2
  • H. J. Iland
    • 1
  1. 1.The Kanematsu LaboratoriesRoyal Prince Alfred HospitalCamperdownAustralia
  2. 2.CSIRO Division of Biomolecular EngineeringNorth RydeAustralia

Personalised recommendations