title: Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis.
abstract: The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.
authors.affiliation: Department of Applied Chemistry, National Defense Academy, Hashirimizu 1-10-20, Yokosuka, Kanagawa, 239-8686, Japan, khiratsu@nda.ac.jp.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Hiratsu
authors.forename: Shiori
authors.initials: S
authors.lastname: Shiotani
authors.forename: Kozo
authors.initials: K
authors.lastname: Makino
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Nunoshiba
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods; D032461:Chromosomes, Plant / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D018744:DNA, Plant / Q000032:analysis / Q000187:drug effects; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D016153:Genes, Suppressor; D005822:Genetic Vectors / Q000235:genetics; D042822:Genomic Instability / Q000187:drug effects / Q000235:genetics; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D009153:Mutagens; C096275:supF tRNA; D012343:RNA, Transfer; D005020:Ethyl Methanesulfonate