Matching Items (6)
Filtering by
- All Subjects: Evolution
- Creators: Lynch, Michael
- Status: Published
Description
Many factors are at play within the genome of an organism, contributing to much of the diversity and variation across the tree of life. While the genome is generally encoded by four nucleotides, A, C, T, and G, this code can be expanded. One particular mechanism that we examine in this thesis is modification of bases—more specifically, methylation of Adenine (m6A) within the GATC motif of Escherichia coli. These methylated adenines are especially important in a process called methyl-directed mismatch repair (MMR), a pathway responsible for repairing errors in the DNA sequence produced by replication. In this pathway, methylated adenines identify the parent strand and direct the repair proteins to correct the erroneous base in the daughter strand. While the primary role of methylated adenines at GATC sites is to direct the MMR pathway, this methylation has also been found to affect other processes, such as gene expression, the activity of transposable elements, and the timing of DNA replication. However, in the absence of MMR, the ability of these other processes to maintain adenine methylation and its targets is unknown.
To determine if the disruption of the MMR pathway results in the reduced conservation of methylated adenines as well as an increased tolerance for mutations that result in the loss or gain of new GATC sites, we surveyed individual clones isolated from experimentally evolving wild-type and MMR-deficient (mutL- ;conferring an 150x increase in mutation rate) populations of E. coli with whole-genome sequencing. Initial analysis revealed a lack of mutations affecting methylation sites (GATC tetranucleotides) in wild-type clones. However, the inherent low mutation rates conferred by the wild-type background render this result inconclusive, due to a lack of statistical power, and reveal a need for a more direct measure of changes in methylation status. Thus as a first step to comparative methylomics, we benchmarked four different methylation-calling pipelines on three biological replicates of the wildtype progenitor strain for our evolved populations.
While it is understood that these methylated sites play a role in the MMR pathway, it is not fully understood the full extent of their effect on the genome. Thus the goal of this thesis was to better understand the forces which maintain the genome, specifically concerning m6A within the GATC motif.
To determine if the disruption of the MMR pathway results in the reduced conservation of methylated adenines as well as an increased tolerance for mutations that result in the loss or gain of new GATC sites, we surveyed individual clones isolated from experimentally evolving wild-type and MMR-deficient (mutL- ;conferring an 150x increase in mutation rate) populations of E. coli with whole-genome sequencing. Initial analysis revealed a lack of mutations affecting methylation sites (GATC tetranucleotides) in wild-type clones. However, the inherent low mutation rates conferred by the wild-type background render this result inconclusive, due to a lack of statistical power, and reveal a need for a more direct measure of changes in methylation status. Thus as a first step to comparative methylomics, we benchmarked four different methylation-calling pipelines on three biological replicates of the wildtype progenitor strain for our evolved populations.
While it is understood that these methylated sites play a role in the MMR pathway, it is not fully understood the full extent of their effect on the genome. Thus the goal of this thesis was to better understand the forces which maintain the genome, specifically concerning m6A within the GATC motif.
ContributorsBoyer, Gwyneth (Author) / Lynch, Michael (Thesis director) / Behringer, Megan (Committee member) / Geiler-Samerotte, Kerry (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Hundreds of thousands of people die annually from malaria; a protozoan of the genus Plasmodium is responsible for this mortality. The Plasmodium parasite undergoes several life stages within the mosquito vector, the transition between which require passage across the lumen of the mosquito midgut. It has been observed that in about 15% of parasites that develop ookinetes in the mosquito abdomen, sporozoites never develop in the salivary glands, indicating that passage across the midgut lumen is a significant barrier in parasite development (Gamage-Mendis et al., 1993). We aim to investigate a possible correlation between passage through the midgut lumen and drug-resistance trends in Plasmodium falciparum parasites. This study contains a total of 1024 Anopheles mosquitoes: 187 Anopheles gambiae and 837 Anopheles funestus samples collected in high malaria transmission areas of Mozambique between March and June of 2016. Sanger sequencing will be used to determine the prevalence of known resistance alleles for anti-malarial drugs: chloroquine resistance transporter (pfcrt), multidrug resistance (pfmdr1) gene, dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr). We compare prevalence of resistance between abdomen and head/thorax in order to determine whether drug resistant parasites are disproportionately hindered during their passage through the midgut lumen. A statistically significant difference between resistance alleles in the two studied body sections supports the efficacy of new anti-malarial gene surveillance strategies in areas of high malaria transmission.
ContributorsPhillips, Keeley Isabella (Author) / Huijben, Silvie (Thesis director) / Gile, Gillian (Committee member) / Young, Steven (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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
Description
Phenotypic evolution is an essential topic within the general field of evolution. Theoretically, the outcome of phenotypic evolution may be influenced by factors such as genetic background and the interaction of natural selection and genetic drift. To gain empirical evidence for testing the effects of those factors, we used eight long-term evolved Escherichia coli populations as a model system. These populations differ in terms of genetic background (different mutation rates) as well as bottleneck size (small- and large-magnitude). Specifically, we used a plate reader to measure three growth-related traits: maximum growth rate (umax), carrying capacity (Kc), and lag time (Lt) for 40 clones within each population. For each trait we quantified the change in mean per generation, the change in variance per generation, and the correlation coefficient between pairs of traits. Interestingly, we found that the small and large bottleneck populations of one background displayed clear, distinguishing trends that were not present within the populations of the other background. This leads to the conclusion that the influence of selection and drift on a population’s phenotypic outcomes is itself influenced by the genetic background of that population. Additionally, we found a strong positive correlation between umax and Kc within each of the high-mutation populations that was not consistent with our neutral expectation. However, the other two pairs did not exhibit a similar pattern. Our results provide a novel understanding in the relationship between the evolution of E. coli growth-related phenotypes and the population-genetic environment.
ContributorsGonzales, Jadon (Co-author, Co-author) / Lynch, Michael (Thesis director) / Ho, Wei-Chin (Committee member) / Geiler-Samerotte, Kerry (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Mutation rate is the rate of appearance for mutations to occur in a living organism. Studying and quantifying mutation rates and their evolution is important because mutations are the ultimate source of genetic variation and one of the reasons why evolution occurs. Much of the current research has investigated the mutational rate increase. The evolution of reduced mutation rate, which can be favored by natural selection because the accumulation of too many mutations can be deleterious and result in death, is less studied. Therefore, this study will be focused on antimutators, which are mutations that result in a lowering of the mutation rate. Using Escherichia coli K-12 str. MG1655 as a model system, the effects and reasons for how MMR- background E. coli evolves lower mutation rates were studied. Here we show that the candidate antimutator in dnaE lowers the mutation rate in an experimentally evolved population of E. coli with MMR- background by using a mutation rate assay to demonstrate the difference between populations with and without the antimutator candidate. The results also suggest the importance of an antimutator for populational survival.
ContributorsGraham, Logan (Author) / Ho, Wei-Chin (Thesis director) / Lynch, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05
Description
Different populations of evolved E.coli and their ancestors were grown in a variety of single amino acid environments to determine their ability to use that amino acid as a carbon source. Some evolved lines were able to grow in amino acids that their ancestors weren't able to. The source of this change in amino acid growth was investigated by testing uptake, searching for candidate mutations, and comparing growth rates of populations with and without certain mutations.
ContributorsKing, Lily (Author) / Ho, Wei-Chin (Thesis director) / Lynch, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2022-05