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Open AccessArticle

Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

1
Department of Chromosome Biology, University of Vienna, 1030 Vienna, Austria
2
Department of Molecular, Cellular and Developmental Biology, University of California at Santa Barbara, Santa Barbara, CA 93106, USA
3
Department of Biodiversity Science, Division of Biological Resource Science, Graduate School of Agricultural Science, Tohoku University, Oosaki 989-6711, Japan
4
Department of Biology, York University, Toronto, ON M3J 1P3, Canada
*
Author to whom correspondence should be addressed.
Current address: Department of Neurology, Neuromuscular Division, Washington University School of Medicine, St Louis, MO 63110, USA.
Genes 2018, 9(4), 179; https://doi.org/10.3390/genes9040179
Received: 19 February 2018 / Revised: 16 March 2018 / Accepted: 20 March 2018 / Published: 23 March 2018
(This article belongs to the Section Microbial Genetics and Genomics)
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 the mechanism of 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. View Full-Text
Keywords: 6-methylpurine; adenine phosphoribosyltransferase; mutation; selection marker; Tetrahymena thermophila 6-methylpurine; adenine phosphoribosyltransferase; mutation; selection marker; Tetrahymena thermophila
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Akematsu, T.; Findlay, A.; Fukuda, Y.; Pearlman, R.E.; Loidl, J.; Orias, E.; P. Hamilton, E. Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena. Genes 2018, 9, 179.

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