Akematsu Takahiko, Findlay Andrew, Fukuda Yasuhiro, Pearlman Ronald E, Loidl Josef, Orias Eduardo, P Hamilton Eileen
Department of Chromosome Biology, University of Vienna, 1030 Vienna, Austria.
Department of Molecular, Cellular and Developmental Biology, University of California at Santa Barbara, Santa Barbara, CA 93106, USA.
Genes (Basel). 2018 Mar 23;9(4):179. doi: 10.3390/genes9040179.
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 that are resistant to 6mp were isolated in 1974, but the mechanism of resistance has remained unknown. To investigate 6mp resistance in , we created 6mp-resistant strains and identified a mutation in the APRTase genomic locus () that is responsible for 6mp resistance. While overexpression of the mutated allele in 6mp-sensitive cells did not confer resistance to 6mp, reduced wild-type expression resulted in a significant decrease in sensitivity to 6mp. Knocking out or reducing the expression of 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.
6-甲基嘌呤（6mp）是腺嘌呤的一种毒性类似物，可抑制RNA和蛋白质合成，并干扰由腺嘌呤磷酸核糖基转移酶（APRTase）介导的腺嘌呤补救途径。1974年分离出了对6mp具有抗性的纤毛原生生物突变体，但其抗性机制仍不清楚。为了研究对6mp的抗性，我们构建了对6mp具有抗性的菌株，并在APRTase基因组位点（）中鉴定出一个导致对6mp产生抗性的突变。虽然在对6mp敏感的细胞中突变等位基因的过表达并未赋予对6mp的抗性，但野生型表达的降低导致对6mp的敏感性显著降低。通过RNA干扰（RNAi）敲除或降低的表达并不影响细胞的强劲生长，这表明腺嘌呤补救途径是多余的，或者从头合成途径为细胞存活提供了足够的一磷酸腺苷。我们还通过构建对6mp和巴龙霉素具有抗性的双突变体，探索了6mp抗性是否可以用作一种新型的诱导选择标记。虽然对6mp和巴龙霉素具有抗性的双突变体在基于RNAi的系统中确实表达了荧光蛋白，但在6mp抗性能够用作有效的诱导选择标记之前该系统需要优化。
