Reversion-Reporter Transgenes to Analyze All Six Base-Substitution Pathways in Arabidopsis

Abstract

Abstract To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS  + only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS– proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.