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Analysis of CAD Gene Amplification Using a Combined Approach of Molecular Genetics and Cytogenetics

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Eukaryotic Cell Cultures

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 172))

Abstract

CAD2 is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA,3 a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.

This work was supported by research grant numbers GH27754, CA 13884 and CA14195 from the NIH D.H.H.S. B.R.S.V. was supported by a NATO fellowship and by the French CNRS. J.M. was supported by NIH predoctoral training grant #PHS CA 09345.

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Authors and Affiliations

  1. Tumor Virology Laboratory, The Salk Institution for Biological Studies, La Jolla, California, 92037, USA

    Geoffrey M. Wahl, Virginia Allen, Suzanne Delbruck, Walter Eckhart, Judy Meinkoth, Bruno Robert de Saint Vincent & Louise Vitto

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  1. Geoffrey M. Wahl
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  2. Virginia Allen
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  3. Suzanne Delbruck
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  4. Walter Eckhart
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  5. Judy Meinkoth
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  6. Bruno Robert de Saint Vincent
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  7. Louise Vitto
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Editors and Affiliations

  1. Department of Microbiology, University of Alabama in Birmingham, Birmingham, Alabama, USA

    Ronald T. Acton  & J. Daniel Lynn  & 

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© 1984 Plenum Press, New York

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Wahl, G.M. et al. (1984). Analysis of CAD Gene Amplification Using a Combined Approach of Molecular Genetics and Cytogenetics. In: Acton, R.T., Daniel Lynn, J. (eds) Eukaryotic Cell Cultures. Advances in Experimental Medicine and Biology, vol 172. Springer, New York, NY. https://doi.org/10.1007/978-1-4615-9376-8_19

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  • DOI: https://doi.org/10.1007/978-1-4615-9376-8_19

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