Development of a new system for detecting mutation in higher plants and specificity of mutational spectra

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Reactive oxygen species, ionizing radiation, UV and various chemicals damage DNA and induce retardation of gene expression, replication and mutation. In plants continuously exposed to UV contained in solar radiation, various types of damages should be produced in genetic substances. These damages has been known to induce mutation. A new system was developed for detection and analysis of mutations occurring on chromosomal DNA in Arabidopsis. The plasmid pML4 carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plant DNA exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to detect mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E.coli cells resistant to streptomycin. We successfully established the assay system of mutation occurring in higher plants. We investigated the mutational specificity in Arabidopsis treated with ethtylmethanesulfonate (EMS).[Results]Mutant frequency of EMS treated plants was 20-fold higher than background mutation. Frameshifts, deletions and sequence substitutions were observed in background mutation. Sequence substitutions have not detected in mouse and fish. GC to AT transitions dominated in EMS-induced mutation. Almost all 3'-purine flanking to target G was A, which was different from animals.