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Description
Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer. An understanding of the variables which impact mutation rates and their estimation is necessary to place mutation rate estimates in their proper contexts. To better understand mutation rate estimates, this research investigates the impact of temperature upon transcription rate error estimates; the impact of growing cells in liquid culture vs. on agar plates; the impact of many in vitro variables upon the estimation of deoxyribonucleic acid (DNA) mutation rates from a single sample; and the mutational hazard induced by expressing clustered regularly interspaced short palindromic repeat (CRISPR) proteins in yeast. This research finds that many of the variables tested did not significantly alter the estimation of mutation rates, strengthening the claims of previous mutation rate estimates across the tree of life by diverse experimental approaches. However, it is clear that sonication is a mutagen of DNA, part of an effort which has reduced the sequencing error rate of circle-seq by over 1,000-fold. This research also demonstrates that growth in liquid culture modestly skews the mutation spectrum of MMR- Escherichia coli, though it does not significantly impact the overall mutation rate. Finally, this research demonstrates a modest mutational hazard of expressing Cas9 and similar CRISPR proteins in yeast cells at an un-targeted genomic locus, though it is possible the indel rate has been increased by an order of magnitude.
ContributorsBaehr, Stephan (Author) / Lynch, Michael (Thesis advisor) / Geiler-Samerotte, Kerry (Committee member) / Mangone, Marco (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2023
Description
Regulation of transcription initiation is a critical factor in the emergence of diverse biological phenotypes, including the development of multiple cell types from a single genotype, the ability of organisms to respond to environmental cues, and the rise of heritable diseases. Transcription initiation is regulated in large part by promoter regions of DNA. The identification and characterization of cis-regulatory regions, and understanding how these sequences differ across species, is a question of interest in evolution. To address this topic, I used the model organism Daphnia pulex, a well-characterized microcrustacean with an annotated genome sequence and selected a distribution of well-defined populations geographically located throughout the Midwestern US, Oregon, and Canada. Using isolated total RNA from adult, female Daphnia originating from the selected populations as well as a related taxon, Daphnia pulicaria (200,000 years diverged from D. pulex), I identified an average of over 14,000 (n=14,471) promoter regions using a novel transcription start site (TSS) profiling method, STRIPE-seq. Through the identification of sequence architecture, promoter class, conservation, and transcription start region (TSR) width, of cis-regulatory regions across the aforementioned Daphnia populations, I constructed a system for the study of promoter evolution, enabling a robust interpretation of promoter evolution in the context of the population-genetic environment. The methodology presented, coupled with the generated dataset, provides a foundation for the study of the evolution of promoters across both species and populations.
ContributorsSnyder, Shannon (Author) / Lynch, Michael (Thesis advisor) / Harris, Robin (Committee member) / Raborn, Randolph T (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2020