Biochemical Genetics

, Volume 30, Issue 11–12, pp 635–648 | Cite as

Molecular and biochemical elucidation of a cellular phenotype characterized by adenine analogue resistance in the presence of high levels of adenine phosphoribosyltransferase activity

  • Nada H. Khattar
  • Gregory E. Cooper
  • Debra L. DiMartino
  • Peggy L. Bishop
  • Mitchell S. Turker
Article
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Abstract

A mouse embryonal carcinoma cell line isolated for resistance to the adenine analogue 2,6-diaminopurine (DAP) was found to have near-wild-type levels of adenine phosphoribosyltransferase (APRT) activity in a cell-free assay. This DAP-resistant (DAPr) cell line, termed H29D1, also exhibited near-wild-type levels of adenine accumulation and the ability to grow in medium containing azaserine and adenine. Growth in this medium requires high levels of intracellular APRT activity. Using the polymerase chain reaction (PCR) and the dideoxy chain termination sequencing technique, an A → G transition was discovered in exon 3 of theaprt gene in H29D1. This mutation resulted in an Arg-to-Gln change at amino acid 87 of the APRT protein that, in turn, resulted in a decreased affinity for adenine. An increased sensitivity of APRT to inhibition by AMP was observed when comparing H29D1 to P19, the parental cell line. Using a transgene containing the A → G mutation, we demonstrated that this mutation is responsible for the biochemical and cellular phenotypes observed for the H29D1 cell line. The approach used in this study provides a definitive method for linking a mutation to a specific cellular phenotype.

Key words

mouse adenine phosphoribosyltransferase base-pair substitution polymerase chain reaction structure-function relationship 
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Copyright information

© Plenum Publishing Corporation 1992

Authors and Affiliations

  • Nada H. Khattar
    • 1
  • Gregory E. Cooper
    • 1
  • Debra L. DiMartino
    • 2
  • Peggy L. Bishop
    • 2
  • Mitchell S. Turker
    • 1
    • 2
    • 3
  1. 1.Department of Microbiology and ImmunologyUniversity of Kentucky College of MedicineLexington
  2. 2.Department of PathologyUniversity of Kentucky College of MedicineLexington
  3. 3.The Lucille Parker Markey Cancer CenterLexington

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