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- All Subjects: Molecular Biology
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
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
The branching dynamics and navigation of filamentous fungi that have an apical vesical crescent (AVC) are poorly understood. Here, Rhizopus oryzae (Mucoromycota), which has an AVC, is compared to Neurospora crassa (Ascomycota), which has a Spitzenkörper (Spk), as they navigated microfluidic maze environments varying in pattern. The different maze patterns (diamonds, squares, and chevrons) presented increasing angles of impact, and degrees of obstruction. This investigation addressed questions regarding advantages or disadvantages that a Spk or AVC may provide in hyphal growth. All branching phenomena were compared to the regular branching of unobstructed growth to determine obstacle induced branching. Neurospora crassa generated more branches per impact amongst all three maze types and was unable to complete the chevron maze types. Rhizopus oryzae generated less branches per impact but was able to complete every maze type. The greatest difference in branch formation was seen in the chevron maze design where N. crassa generated a greater number than R. oryzae. Neurospora crassa exhibited a hyperbranching response in the chevron mazes not seen in R. oryzae. Closer inspection of the hyperbranching events revealed that they were composed of initial branching events followed by secondary and tertiary branching events. The directional memory of N. crassa was also observed, and was a characteristic of R. oryzae. While the branching dynamics and navigation of N. crassa and R. oryzae were different, and N. crassa exhibited branching and navigational phenomenon that R. oryzae did not, R. oryzae seemingly had the advantage with its use of an AVC over N. crassa, as it was able to complete every maze type, which N. crassa was unable to do.
ContributorsGonzalez, Benjamin (Author) / Roberson, Robert W (Thesis advisor) / Baluch, Debra P (Thesis advisor) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2021
Description
Single-cell DNA sequencing (scDNA-seq) can identify genetic differencesbetween individual cells and has broad applications in studying biology. For example, because scDNA-seq preserves haplotypes, it enables the addition of information about the fitness of different combinations of mutations into studies that quantify the fitness of individual mutations. However, it requires separating cells manually or using machinery, which is time-consuming and costly as every cell requires a separate reaction. Thus, most studies are limited to a few hundred cells, and scaling up is expensive and challenging. This problem also makes it difficult to multiplex samples or to study multiple sample types in the same experiment. To solve these problems, I introduce a novel method for sequencing DNA in heterogeneous cell populations by using the cell itself as a container for sequencing reactions, eliminating the need to isolate individual cells. The method involves diffusing DNA polymerase and barcoded primers into intact cells and amplifying its DNA Intracellularly. To ensure that DNA from each cell can be uniquely identified, I use combinatorial barcoding, which assigns a specific barcode to each cell using a unique combination of non-unique nucleotide block sequences. This allows for the pooling of cells, making the method multiplexable and enabling the analysis of dozens of samples containing thousands of cells. The method is flexible and allows for targeted sequencing of a region of interest and whole genome sequencing. I optimize the method for various organisms and applications so it can be made accessible to a wide range of research groups.
ContributorsCrossland, Parker Ella (Author) / Geiler-Samerotte, Kerry (Thesis advisor) / Wideman, Jeremy (Committee member) / Brettner, Leandra (Committee member) / Arizona State University (Publisher)
Created2024
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