The Effect of Overexpressed AtOgg1 on Spontaneous Guanine-to-Thymine Mutation Colin Tominey Dr. John Hays Oregon State University June 27, 2008 Background • All organisms experience a certain number of mutations due to chemical and environmental interactions with their DNA. • Point mutations have two general causes: – Chemical modification of a DNA base – DNA replication error Arabidopsis as a Model Organism • Arabidopsis thaliana is often used as a model differentiating organism. • Advantages: – Short generation times (~6 weeks) – Small, well-documented genome – Transgenic varieties are easily created – Survives multiple gene knockouts Arabidopsis and Mutation Research • Insertion of modified GUS genes into Arabidopsis created mutation-reporting plants. • Each line is designed to report one class of point mutation (e.g., G→T) • Reversion of the designated base reactivates the GUS gene and produces blue spots on that plant. GUS Mutation Reporters wt GUS Codon 11: Active Protein -C G T-G A A-A T C-G C A-C T T-T A GGUS G→T Reporter Codon 11: Inactive Protein -C G T-G C A-A T C-G C A-C G T-T A G• When a mutation causes the reporter codon to revert to the wt codon, active protein is produced and plant cells are stained blue. Unexpected Observation • Spontaneous reversion in the GUS G T lines was much higher than that in other GUS plants: Spontaneous Reversions Reversion Type Spontaneous Reversions (Events / Plant) G→T 0.2 – 2.1 C→T 0.007 – 0.03 T→C 0 – 0.01 T→G 0 – 0.03 G→C 0.03-0.04 Potential Sources of G → T Mutation • 8-oxoguanine has been implicated in G → T mutation: – Pairs strongly with adenine, causing mutation during replication – Generated by reactive oxygen species attacking guanine in the DNA strand GUS G→T Mutation Reporter Reversion -GCA-CGTO -GCA-CGT- Top strand retains the pre-existing codon. REPLICATION* X O 8 O- Bottom strand forms a pre-mutagenic lesion. -GAA-CTT- REPLICATION* Top strand results in fixation of mutation (reversion to wt) and production of active GUS protein. -GAA-CGTO X O 8 * Assumes replication without repair of the lesion. Defending DNA • AtOgg1, AtFpg, and AtMyh all code for proteins that protect Arabidopsis. • Many bacteria and eukaryotes (including humans) have these same kinds of proteins. • Are these defenses sufficient? Hypothesis • High levels of 8-oxoguanine have overwhelmed the plant’s natural defenses (OGG1, FPG, and MYH), thus causing the unexpectedly-high rate of spontaneous G → T mutation. Prediction • Plants with enhanced AtOgg1 expression will show significantly less spontaneous G → T mutation than their siblings with normal AtOgg1 expression. The Experiment • Create a line of Arabidopsis plants that overexpress the AtOgg1 gene. • Cross these mutants with existing GUS G → T reporter lines. • Compare spontaneous GUS reversion in new mutants with non-overexpressing siblings. Acknowledgments • • • • • • • Howard Hughes Medical Institute Oregon State University URISC Dr. John Hays Dr. Marc Curtis Dr. Stephanie Bollmann Peter Hoffman Dr. Kevin Ahern