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Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer

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Somatic Cell and Molecular Genetics

Abstract

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.

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  1. T. Michael Underhill

    Present address: Department of Microbiology, Duke University Medical Center, Box 3020, 27710, Durham, North Carolina

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Western Ontario, N6A 5C1, London, Ontario, Canada

    T. Michael Underhill, Frederick M. R. Williams, Richard C. Murray & Wayne F. Flintoff

Authors
  1. T. Michael Underhill
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  2. Frederick M. R. Williams
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  3. Richard C. Murray
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  4. Wayne F. Flintoff
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Underhill, T.M., Williams, F.M.R., Murray, R.C. et al. Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer. Somat Cell Mol Genet 18, 337–349 (1992). https://doi.org/10.1007/BF01235757

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  • DOI: https://doi.org/10.1007/BF01235757

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