Advertisement

Controllable Expression of an E. Coli Amidophosphoribosyltransferase (ATase) Gene in ATase-Deficient Mammalian Fibroblasts—a Basic Model for Gene Therapy

  • Mitsuo Itakura
  • Takashi Yamaoka
  • Hiroko Yoshikawa
  • Kamejiro Yamashita
  • Richard Sabina
  • Howard Zalkin
  • Edward Holmes
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 253A)

Abstract

Amidophosphoribosyltransferase (ATase) (E.C.2.1.2.14) is presumably a rate-limiting enzyme in de novo purine synthetic pathway under a skillful regulatory system, although 5-phosphoribosyl 1-pyrophosphate (PRPP) synthetase is another possibility. The present study was designed to investigate the rate-limiting quality of ATase by artificially controlling expression of its activity, and to test the complementality of ATase-deficiency with a foreign ATase gene in mammalian cells. The experimental scheme, as in Fig. 1, was composed of production of a recombinant plasmid between an E. coli ATase gene and a glucocorticoid-responsive promoter and its transfection into a strain of Chinese hamster ovary fibroblasts which is deficient in ATase (CHO ade-A). Transfectants were cultured in the presence or absence of glucocorticoid, and were examined as to the complementality of de novo purine and DNA synthesis in purine-free medium with dexamethasone (DEX). The expression of E. coli ATase was also examined by northern, western analysis, and ATase activity.

Keywords

Herpes Zoster Thymidine Kinase Scintillation Counter Northern Analysis Western Analysis 
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.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    L.J. Messenger, and H. Zalkin, Glutamine phosphoribosylpyrophosphate amidotransferase from Eschrichia coli, Purification and properties. J. Biol. Chem. 254: 3382 (1978).Google Scholar
  2. 2.
    J.Y. Tso, H. Zalkin, M. van Cleemput, C. Yanofsky, and J.M. Smith, Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. 257: 3525 (1981).Google Scholar
  3. 3.
    V.L. Chandler, B.A. Maler, and K.R. Yamamoto, DNA sequence bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell 33: 488 (1983).CrossRefGoogle Scholar
  4. 4.
    M. Itakura and E.W. Holmes, Human amidophosphoribosyltransferase, an oxygen-sensitive iron-sulfur protein. J. Biol. Chem. 254: 333 (1979).PubMedGoogle Scholar
  5. 5.
    Gel electrophoresis, Chapt. 5, pp149-186, in Molecular Cloning, eds by T. Maniatis, E.F. Pritsch, and J. Sambrook, (1982).Google Scholar
  6. 6.
    H. Towbin, T. Staehelin, and J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350 (1979).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • Mitsuo Itakura
    • 1
  • Takashi Yamaoka
    • 1
  • Hiroko Yoshikawa
    • 1
  • Kamejiro Yamashita
    • 1
  • Richard Sabina
    • 2
  • Howard Zalkin
    • 3
  • Edward Holmes
    • 2
  1. 1.Inst. Clin. Med.Univ. TsukubaTsukuba-city, Ibaraki 305Japan
  2. 2.Dept. Med.Duke Univ. Med. Ctr.USA
  3. 3.Dept. Biochem.Purdue Univ.USA

Personalised recommendations