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.
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This work was supported by NIH Grant AG08199. G.E.C. was supported by a College of Medicine Dean's fellowship. The fluoroadenine used in this study was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute.
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Khattar, N.H., Cooper, G.E., DiMartino, D.L. et al. Molecular and biochemical elucidation of a cellular phenotype characterized by adenine analogue resistance in the presence of high levels of adenine phosphoribosyltransferase activity. Biochem Genet 30, 635–648 (1992). https://doi.org/10.1007/PL00020523
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DOI: https://doi.org/10.1007/PL00020523