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

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 mech...

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Autores principales: Akematsu, Takahiko, Findlay, Andrew, Fukuda, Yasuhiro, E. Pearlman, Ronald, Loidl, Josef, Orias, Eduardo, P. Hamilton, Eileen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924521/
https://www.ncbi.nlm.nih.gov/pubmed/29570682
http://dx.doi.org/10.3390/genes9040179
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author Akematsu, Takahiko
Findlay, Andrew
Fukuda, Yasuhiro
E. Pearlman, Ronald
Loidl, Josef
Orias, Eduardo
P. Hamilton, Eileen
author_facet Akematsu, Takahiko
Findlay, Andrew
Fukuda, Yasuhiro
E. Pearlman, Ronald
Loidl, Josef
Orias, Eduardo
P. Hamilton, Eileen
author_sort Akematsu, Takahiko
collection PubMed
description 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.
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spelling pubmed-59245212018-05-03 Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena Akematsu, Takahiko Findlay, Andrew Fukuda, Yasuhiro E. Pearlman, Ronald Loidl, Josef Orias, Eduardo P. Hamilton, Eileen Genes (Basel) Article 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. MDPI 2018-03-23 /pmc/articles/PMC5924521/ /pubmed/29570682 http://dx.doi.org/10.3390/genes9040179 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Akematsu, Takahiko
Findlay, Andrew
Fukuda, Yasuhiro
E. Pearlman, Ronald
Loidl, Josef
Orias, Eduardo
P. Hamilton, Eileen
Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title_full Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title_fullStr Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title_full_unstemmed Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title_short Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena
title_sort resistance to 6-methylpurine is conferred by defective adenine phosphoribosyltransferase in tetrahymena
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924521/
https://www.ncbi.nlm.nih.gov/pubmed/29570682
http://dx.doi.org/10.3390/genes9040179
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