An In Vitro Transcription System from BmN Cells of the Silkworm, Bombyx mori

  • Hiroshi Sakurai
  • Susumu Izumi
  • Shiro Tomino
Part of the Methods in Molecular Biology book series (MIMB, volume 37)


Eukaryotic RNA polymerase II requires a set of protein factors for the accurate initiation of gene transcription (1). Whole-cell and nuclear extracts containing these components have been prepared from cultured cells and used to execute faithful transcription initiation on the DNA templates (2, 3, 4, 5, 6, 7). Transcriptionally active extracts have also been obtained from embryos (5, 8) and differentiated tissues (5, 9, 10). In order to facilitate studies of class II gene regulation in Lepidopteran insects, we developed a cell-free system from BmN cells, a cell line established from an ovarian tissue of the silkworm, Bombyx mori (7, 11). The essential features of our in vitro transcription system may be summarized as follows:


Nuclear Extract Dounce Homogenizer Primer Extension Analysis Calf Intestinal Alkaline Phosphatase Core Promoter Element 
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.


  1. 1.
    Sawadogo, M. and Sentenac, A. (1990) RNA polymerase B (II) and general transcription factors. Annu. Rev. Biochem. 59, 711–754.PubMedCrossRefGoogle Scholar
  2. 2.
    Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. USA 77, 3855–3859.PubMedCrossRefGoogle Scholar
  3. 3.
    Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription mitiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 5, 1475–1489.CrossRefGoogle Scholar
  4. 4.
    Parker, C. S. and Topol, J. (1984) A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity. Cell 36, 357–369.PubMedCrossRefGoogle Scholar
  5. 5.
    Suzuki, Y., Tsuda, M., Takiya, S., Hirose, S., Suzuki, E., Kameda, M., and Ninaki, O. (1986) Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems. Proc. Natl. Acad. Sci. USA 83, 9522–9526.PubMedCrossRefGoogle Scholar
  6. 6.
    Kobayashi, A., Hirai, H., Kubo, T., Ueno, K., Nakanishi, Y., and Natori, S. (1989) Cloning and in vitro transcription of Sarcophaga lectin gene. Biochim. Biophys. Acta 1009, 244–250.PubMedGoogle Scholar
  7. 7.
    Sakurai, H., Izumi, S., and Tomino, S. (1990) In vitro transcription of the plasma protein genes of Bombyx mori. Biochim. Biophys. Acta 1087, 18–24.PubMedGoogle Scholar
  8. 8.
    Heberlein, U. and Tjian, R. (1988) Temporal pattern of alcohol dehydrogenase gene transcription reproduced by Drosophila stage-specific embryonic extracts. Nature 331, 410–415.PubMedCrossRefGoogle Scholar
  9. 9.
    Gorski, K., Carneiro, M., and Schibler, U. (1986) Tissue-specific in vitro transcription from the mouse albumin promoter. Cell 47, 767–776.PubMedCrossRefGoogle Scholar
  10. 10.
    Conaway, J. W., Bond, M. W., and Conaway, R.C. (1987) An RNA polymerase II transcription system from rat liver: purification of an essential component. J. Biol. Chem. 262, 8293–8297.PubMedGoogle Scholar
  11. 11.
    Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y, and Furusawa, M. (1985) Production of human α-interferon in silkworm using a baculovirus vector. Nature 315, 592–594.PubMedCrossRefGoogle Scholar
  12. 12.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  13. 13.
    Maniatis, T., Goodbourn, S., and Fischer, J. A. (1987) Regulation of inducible and tissue-specific gene expression. Science 236, 1237–1245.PubMedCrossRefGoogle Scholar
  14. 14.
    Hirose, S., Tsuda, M., and Suzuki, Y. (1985) Enhanced transcription of fibroin gene in vitro on covalently closed circular templates. J. Biol. Chem. 260, 10,557–10,562.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Hiroshi Sakurai
    • 1
  • Susumu Izumi
    • 1
  • Shiro Tomino
    • 1
  1. 1.Department of BiologyTokyo Metropolitan UniversityTokyoJapan

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