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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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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. |
format | Online Article Text |
id | pubmed-5924521 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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