Tarailo-Graovac, MajaStuart, Amanda Rhae2022-09-262022-09-262022-09Stuart, A. R. (2022). Investigating multiple modifier network candidates as suppressors of zyg-1/PLK4 in C. elegans (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115287Genetic modifiers are second-site alleles that affect the function or regulation of a primary allele to either suppress or enhance the associated phenotype. The traditional methods of identifying and studying these modifying interactions have been useful for the consistent elucidation of single modifier activities, which has expanded the known single gene interactions significantly. However, these methods have been limited in their capability to identify true cases of modifier networks within a genome because of the use of backcrossing to minimize the regions of the genome to be examined for modifiers. By limiting our knowledge of multiple modifier networks, we lack knowledge on the intrinsic complexities of the genome, and how the larger genomic context may be influencing phenotypic outcomes. This thesis explores the use of high-throughput mutagenesis-based suppressor screen methods of a temperature-sensitive, lethal C. elegans allele, zyg-1(it25); these methods include whole genome sequencing and bioinformatic analyses without backcrossing steps, followed by candidate modifier validation using CRISPR/Cas9 methods to study potential multiple modifier networks. Our results show that ~20% of all mutagenized strains have more than one candidate modifier identified after bioinformatic analysis, and CRISPR/Cas9 validation of the candidate networks derived from four mutagenized strains showed that 50% of tested strains carried true multiple modifier networks. Six individual modifier alleles of zyg-1(it25) were validated while testing the candidate networks, including a novel suppressor. Furthermore, study of the candidate alleles in a wild-type background showed that all modifying variants induced their own phenotype, reducing the population viability of these strains. Finally, this study explores the mechanism for suppression of zyg-1(it25) from the validated modifiers, most of which are components of the Anaphase Promoting Complex; all modifying variants show delays of the cell cycle, particularly of anaphase-onset, indicating that increased mitotic time is allowing embryos to overcome deficiencies of ZYG-1. With the ZYG-1 human ortholog, PLK4, being implicated in symptomatic microcephaly, elucidation of the modifier networks and phenotype-altering interaction may lead to further understanding of the mechanisms that cause developmental microcephaly and can have expanded applications to other genetic diseases.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.ModifiersGenomicsModifier NetworksGeneticsPhysics--Molecularmaster thesis