Graduate Thesis Or Dissertation
 

Reversion reporters in Arabidopsis thaliana to detect all six base substitution pathways

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  • Maintaining genome integrity is essential for an organism, as mutation accumulation can lead to cancer, reduced fitness, and heritable diseases in offspring. Therefore the study of mutations, how they are induced, and how they are prevented is vital. Biomonitor systems are useful for understanding the relevant biological effects of a given mutagen, and depending on the system, can even provide information on specific molecular changes induced by the mutagen. Plants are ideal biomonitors, as a sedentary lifestyle allows measurement of mutagens in air, soil, and water, even at low doses. We constructed mutation reporters in Arabidopsis designed to restore β- glucuronidase (GUS) activity through one of six base substitution reversions. All six mutant constructs contained inactivating base substitutions in the same codon, which minimized sequence context effects. An AcV5 epitope tag sequence was fused to the 3' end of GUS to allow detection of the inactive protein and selection of sublines based on levels of GUS protein expression. Initial characterization of these reversion reporters with or without UV-C treatment and exposure to heavy metal ions (Cd²⁺ and Zn²⁺) supports the ability of the lines to measure different mutations. UV-C radiation induced T to C and C to T reversions, as well as T to G and T to A to a lesser extent. Heavy-metal-ion-mutation induction was inconsistent, showing variable increases in G to T and G to C, and no induction of T to C reversion. Of key interest is the G to T reporter line, as this is the first such reporter in higher eukaryotes. This line showed a large increase in spontaneous mutation as well as potential germinal mutations when compared to the other reporter lines, most likely due to endogenous oxidative damage (i.e. 8-oxoguanine). Further experiments to test the reporter lines with various mutagens as well as ways to improve the ability of the reporters to detect mutation are discussed.
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