Random mutagenesis of P. flaciparum by ENU

Research Project

Project/Area Number	26670199
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Parasitology (including sanitary zoology)
Research Institution	Mie University
Principal Investigator	IWANAGA SHIROH 三重大学, 医学(系)研究科(研究院), 准教授 (20314510)
Project Period (FY)	2014-04-01 – 2016-03-31
Project Status	Completed (Fiscal Year 2015)
Budget Amount *help	¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000) Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000) Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Keywords	マラリア / ENU / 感染症
Outline of Final Research Achievements	N-ethyl-N-nitrosourea (ENU) is a potent mutagen and is widely used for random mutagenesis. In the present study, we utilized it for generating the parasite population, whose genomic sequence were randomly mutated, and selected the mefloquine-resistant parasites from them. The obtained mefloquine-resistant parasites exhibited 3 folds stronger resistance than the wild-type parasite. Genomic sequence analysis showed that approximately 2,000 mutations were introduced by ENU treatment. To identify the drug resistant gene, genomic DNA library of mefloquine-resistant parasite was directly generated in drug sensitive parasites using P. falciparum centromere plasmid and the generated library was treated with mefloquine. After three rounds of treatments, three independent parasite clones, which acquired newly mefloquine resistance, were obtained. The centromere plasmids are being recovered from those selected parasites and the drug resistant gene is being identified now.