Date of this Version
McCoy, R. (2020). Inheritability of ciprofloxacin-induced mitochondrial mutations from parental to offspring generation using quantitative polymerase chain reaction. Undergraduate Honors Thesis. University of Nebraska - Lincoln.
In all eukaryotes, mitochondria are known as the powerhouse of the cell (Siekevitz, 1957). In plants, however, their mitochondrial genome is especially strange. Plant mitochondrial genomes are extremely large and contain both linear and circular subgenomic DNA fragments. Plant mitochondrial genomes undergo a significant amount of mutations in the form of rearrangements. However, it is not known how often these rearrangements are inherited by the next generation. It is thought that plant cells that are still dividing have higher rates of DNA repair, such as double-strand break repair, to ensure the quality of that plant lineage. As follows, it is expected that the mutations accumulate in plant cells not destined to produce offspring. This project investigated the effects of ciprofloxacin, a chemical that induces DNA double-strand breaks, on young and old Arabidopsis thaliana (A. thaliana) leaves to test our prediction that mutations accumulate in old plant cells treated with ciprofloxacin. Ciprofloxacin is antibiotic that attacks DNA gyrase, a critical enzyme in DNA replication. DNA gyrase is a topoisomerase that underwinds DNA so that DNA helicase can unwind the DNA for successful replication. When ciprofloxacin is used it renders DNA gyrase nonfunctional and results in tension in the DNA strand, which leads to double-strand breaks. It is likely that double-strand breaks lead to increased recombination because of loose ends. Quantitative polymerase chain reaction (qPCR) was used to measure recombination due to double-strand breaks. A. thaliana grown on 0.5 μM ciprofloxacin plus MSSA plates did not result in more recombination than A. thaliana grown on MSSA only plates, nor was there an effect of leaf age on recombination. This could be due to using too low of a concentration of ciprofloxacin such that the DNA repair mechanisms were sufficient to repair double-strand breaks without resulting in recombination events. Future work will include growing A. thaliana on increasing concentrations of ciprofloxacin plus MSSA plates (0.75 μM, 1 μM, 1.25 μM and 1.5 μM) to determine which plate results in sufficient damage to induce recombination. qPCR will be used to look at the recombinants in comparison to the parentals of A. thaliana on MSSA plus various concentrations of ciprofloxacin and MSSA only plates.