{"id":{"repo_id":"calgary","oai_identifier":"oai:ucalgary.scholaris.ca:1880/115287"},"canonical_url":"https://search.dev.ndltd.org/etd/calgary/oai:ucalgary.scholaris.ca:1880/115287","repository":{"repo_id":"calgary","name":"University of Calgary","base_url":"https://ucalgary.scholaris.ca/server/oai/request"},"display":{"title":"Investigating Multiple Modifier Network Candidates as Suppressors of zyg-1/PLK4 in C. elegans","abstract":"Genetic 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.","abstract_html":"Genetic 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.","abstract_has_math":false,"creators":["Stuart, Amanda Rhae"],"institution":"Cumming School of Medicine","degree_name":"Master of Science (MSc)","degree_level":null,"degree_discipline":"Medicine – Biochemistry and Molecular Biology","degree_department":null,"school":null,"contributors":[],"advisors":["Tarailo-Graovac, Maja"],"committee_chairs":[],"committee_members":["Shutt, Timothy","Hansen, David Donald"],"year":2022,"date_issued":"2022-09","date_published":"2022-09","updated_at":"2026-07-24T01:30:29Z","subjects":["Modifiers","Genomics","Modifier Networks"],"languages":["eng"],"rights":["University 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."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://dx.doi.org/10.11575/PRISM/40293"],"render_values":[{"text":"https://dx.doi.org/10.11575/PRISM/40293","href":"https://dx.doi.org/10.11575/PRISM/40293","code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/1880/115287","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Tarailo-Graovac, Maja"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Shutt, Timothy","Hansen, David Donald"]},{"key":"dc:creator","label":"Author","values":["Stuart, Amanda Rhae"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-02"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2022-09-26T21:54:05Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2022-09-26T21:54:05Z"]},{"key":"dc:date.issued","label":"Date","values":["2022-09"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Calgary"]},{"key":"dc:type","label":"Dc Type","values":["master thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Medicine – Biochemistry and Molecular Biology"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MSc)"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Calgary"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Modifiers","Genomics","Modifier Networks"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["University of Calgary graduate students retain copyright ownership and moral rights for their thesis. 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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."]},{"key":"dc:title","label":"Title","values":["Investigating Multiple Modifier Network Candidates as Suppressors of zyg-1/PLK4 in C. elegans"]}]}],"canonical_facts":{"dc:contributor.advisor":["Tarailo-Graovac, Maja"],"dc:contributor.committeemember":["Shutt, Timothy","Hansen, David Donald"],"dc:creator":["Stuart, Amanda Rhae"],"dc:date":["2023-02"],"dc:date.accessioned":["2022-09-26T21:54:05Z"],"dc:date.available":["2022-09-26T21:54:05Z"],"dc:date.issued":["2022-09"],"dc:description.abstract":["Genetic 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."],"dc:identifier.doi":["https://dx.doi.org/10.11575/PRISM/40293"],"dc:identifier.uri":["http://hdl.handle.net/1880/115287"],"dc:language.iso":["eng"],"dc:publisher.institution":["University of Calgary"],"dc:rights":["University of Calgary graduate students retain copyright ownership and moral rights for their thesis. 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