Resistance to 6-methylpurine is conferred by defective adenine phosphoribosyltransferase in Tetrahymena

Takahiko Akematsu, Andrew Findlay, Yasuhiro Fukuda, Ronald E. Pearlman, Josef Loidl, Eduardo Orias, Eileen P. Hamilton

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

6-methylpurine (6mp) is a toxic analog of adenine that inhibits RNA and protein synthesis and interferes with adenine salvage mediated by adenine phosphoribosyltransferase (APRTase). Mutants of the ciliated protist Tetrahymena thermophila that are resistant to 6mp were isolated in 1974, but themechanismof resistance has remained unknown. To investigate 6mp resistance in T. thermophila, we created 6mp-resistant strains and identified a mutation in the APRTase genomic locus (APRT1) that is responsible for 6mp resistance. While overexpression of the mutated APRT1 allele in 6mp-sensitive cells did not confer resistance to 6mp, reduced wild-type APRT1 expression resulted in a significant decrease in sensitivity to 6mp. Knocking out or reducing the expression of APRT1 by RNA interference (RNAi) did not affect robust cell growth, which indicates that adenine salvage is redundant or that de novo synthesis pathways provide sufficient adenosine monophosphate for viability. We also explored whether 6mp resistance could be used as a novel inducible selection marker by generating 6mp- and paromomycin-resistant double mutants. While 6mp- and paromomycin-resistant double mutants did express fluorescent proteins in an RNAi-based system, the system requires optimization before 6mp resistance can be used as an effective inducible selection marker.

Original languageEnglish
Article number179
JournalGenes
Volume9
Issue number4
DOIs
Publication statusPublished - 2018 Apr

Keywords

  • 6-methylpurine
  • Adenine phosphoribosyltransferase
  • Mutation
  • Selection marker
  • Tetrahymena thermophila

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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  • Cite this

    Akematsu, T., Findlay, A., Fukuda, Y., Pearlman, R. E., Loidl, J., Orias, E., & Hamilton, E. P. (2018). Resistance to 6-methylpurine is conferred by defective adenine phosphoribosyltransferase in Tetrahymena. Genes, 9(4), [179]. https://doi.org/10.3390/genes9040179