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Browsing by Author "Argueso, Juan Lucas, advisor"

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    A yeast-based assay system for the study of environmentally induced copy number variation
    (Colorado State University. Libraries, 2012) Stanton, Jacquelyn Lee, author; Argueso, Juan Lucas, advisor; Legare, Marie, committee member; Stargell, Laurie, committee member
    Multiple studies have shown that in different individuals, specific genomic segments can occur at a variable copy number relative to the reference human genome. Chromosomal rearrangements resulting in Copy Number Variations (CNVs) have long been recognized as contributing factors in carcinogenesis, and more recently in Autism Spectrum Disorders. The molecular mechanisms underlying the formation of CNVs are not completely understood. The goal of this research project was to complete the development of an assay system to study CNV, and to validate it as a tool to investigate the relationship between environmental exposures and CNV formation. We have optimized a CNV detection assay using the budding yeast Saccharomyces cerevisiae as an experimental model system. This CNV reporter contains two yeast genes, SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. This system enables the detection of rare clones containing an amplification of the chromosomal segment containing the reporter by selection in media containing high levels of formaldehyde and copper, allowing the estimation of the rate of CNV formation. Results obtained in diploid cells under basal growth conditions (un-induced / un-exposed) showed that most spontaneous CNV events detected in our system were mediated through non-allelic homologous recombination (NAHR) between dispersed repetitive DNA sequences, mainly Ty1 and Ty2 retrotransposable elements and their LTRs. Another set of repeats involved in NAHR included conserved gene family. Single copy sequences and microhomology motifs were detected in our dataset, but were exceedingly rare. The most abundant classes of CNVs observed involved segmental duplications and non-reciprocal translocations. In order to characterize the effect of environmental factors on CNV, cells were exposed to relatively low doses of three different known mutagens: Hydroxyurea, Methyl Methanesulfonate, and Camptothecin. These exposures resulted in an increase of the CNV rate ranging from 3 to 17 fold over the un-induced cultures. The spectra of chromosomal rearrangements induced by these exposures was analyzed, revealing that not only did exposures result in more chromosomal breaks but often a higher frequency of resulting segmental copies, and allowing further understanding of the CNV mechanisms associated with these exposures.
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    Analysis of the relationship between genomic instability, heterozygosity levels and phenotype in Saccharomyces cerevisiae
    (Colorado State University. Libraries, 2018) Sampaio, Nadia Maria Vieira, author; Argueso, Juan Lucas, advisor; Stargell, Laurie A., committee member; McKay, John K., committee member; Reardon, Kenneth F., committee member
    Understanding the forces that mediate genome evolution is a central problem in genetics, with implications for diverse processes that range from speciation, to biotechnological applications, to human disease. The central theme of my dissertation was the characterization of two forces, genomic instability and natural selection, that significantly impact genome structure by influencing the levels of genomic heterozygosity. While genomic instability processes can act to erode heterozygosity from the genome, natural selection may favor the maintenance of heterozygous alleles in cases where there is a positive correlation between heterozygosity and higher fitness. In Chapter I, I reviewed different types of mitotic mutations that can result in the appearance of tracts of homozygosity in genomes and recent discoveries about the temporal accumulation of such events. I also introduce the concept of heterosis, a phenomenon characterized by a positive correlation between genomic heterozygosity and phenotype in many species, and its potential role in contributing to the long-term maintenance of genomic heterozygosity. In Chapter II, I describe the characterization of a mechanism of systemic genomic instability in yeast that challenges the conventional model of gradual and independent accumulation of mutations. We showed that a subset of mitotic cells within a population experience bursts of genomic instability, which results in multiple independent events of loss-of-heterozygosity (LOH) accumulating over one or a few generations of mitotic cell division. We named this outcome "systemic genomic instability". The occurrence of this phenomenon was initially identified in the heterozygous yeast strain JAY270, and then validated in a conventional laboratory strain background, whose genome is almost fully homozygous. Elevated rates of coincident LOH was also observed in mutant strains incapable of entering meiosis, indicating cryptic initiation of meiotic recombination followed by return-to-growth in a few cells in the population was not responsible for the higher than expected rates of coincident LOH. This finding brings to light a novel and intriguing mechanism of genomic instability in yeast that has relevant parallels to bursts of accumulation of copy number alterations in the human genome, providing a powerful experimental model system to dissect the fundamental mechanisms responsible for the generation of rapid changes in chromosome structure. In Chapter III, we explored the role that genomic heterozygosity plays on the superior industrial traits of the JAY270 strain. In the previous Chapter we showed that mitotic recombination leading to LOH occurs at a high frequency during JAY270's clonal propagation. These LOH events act against the long-term maintenance of genomic heterozygosity, yet about 60% of JAY270's genome has remained heterozygous over time. We hypothesized that specific heterozygous alleles may have a positive impact on the traits of this strain and therefore were maintained through selection. We generated a collection of inbred strains derived from JAY270, and assessed them phenotypically under different growth conditions. Our results demonstrated that genomic heterozygosity indeed has a substantial impact on two important industrial traits of this strain – heat stress tolerance and growth kinetics. We identified several genomic regions potentially associated with those traits and conducted experiments to investigate the bulk contributions of heterozygosity blocks in three specific chromosomes. This study revealed candidate regions containing loci that potentially underlie important industrial traits of JAY270 and details on the extent to which heterozygosity may impact JAY270's genome evolution and phenotype. The combined results of these research projects provide important insights about the role of genomic instability mechanisms and their phenotypic outcomes in determining genome evolution, contributing discoveries that may have important practical implications for diverse fields, including biotechnology, cancer development and evolution, as well as genome sciences.
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    Characterization of biased partner choice in mitotic non-allelic homologous recombination of Saccharomyces cerevisiae
    (Colorado State University. Libraries, 2023) Merriman, Sean, author; Argueso, Juan Lucas, advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Wiese, Claudia, committee member
    Using yeast as a model in which to study copy number variation (CNV)-generating mutations, the J.L. Argueso lab has discovered that a specific region of S. cerevisiae genome (the right arm of chromosome 7; Chr7R) is much more susceptible to sustaining deletions as a translocation recipient than other apparently similar segments of the genome. Further, Chr7R acquires amplifications as a translocation donor less frequently than other chromosomes. To begin unraveling the cause of this unusual behavior, we evaluated the effect of several candidate genes involved in chromatin mobility and sister chromatid cohesion on the mutational spectra involving Chr7R. Our results suggest that regulatory factors of chromatin mobility or sister chromatid cohesion affect the outcomes of HR-mediated repair events at Ch7R. We are hopeful that our findings will open a window into the fundamental cellular processes that are responsible for CNVs found in eukaryotic genomes, and inform translational implications for modeling this class of mutation in cancer.
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    Characterization of modes and kinetics of mutation accumulation in Saccharomyces cerevisiae through the analysis of defined cellular lineages
    (Colorado State University. Libraries, 2024) Stewart, Joseph, author; Argueso, Juan Lucas, advisor; Moreno, Julie, committee member; Regan, Daniel, committee member; Wiese, Claudia, committee member
    In the field of evolution, gradualism is the process of incremental adaptation supported by a slow and random accumulation of mutations that, over time, lead to genetic diversification and fitness gains. Although this Darwinian model is well supported and widely accepted, it cannot always explain the rapid changes seen in some instances such as tumors with extremely high and complex mutation loads. Recent reports in various organisms, including from our group using Saccharomyces cerevisiae, provide evidence for an additional mode of rapid and non-independent accumulation of chromosomal rearrangements. We have used a yeast model to follow the accumulation of structural genomic rearrangements such as loss of heterozygosity (LOH). We found that while chances of a single LOH event happening are very low, two or more LOH tracts co-occurred at rates 25- to 200-fold higher than expected if these events were independent of each other; therefore, the conventional process of slow and independent accumulation of mutations are not sufficient to account for every change in the genome. In the present study, we focused on temporal kinetics of bursts of LOH accumulation in yeast. We developed a hybrid diploid yeast experimental strain that enables identification of LOH event both through counter-selection and visual screening for colony color. This hybrid strain, made from the S288c and SK1 genetic backgrounds, possesses ~55,000 heterozygous SNPs distributed throughout the genome and allows for ease of tracking LOH events through sequencing. The screening approach was used in combination with microcultures (one cell grown for 5 or 6 divisions) in phylogenetic analyses that unambiguously revealed 18 cases where multiple LOH events co-occurred in the same cell division cycle. Collectively, these studies offer support for punctuated bursts of mutation accumulation caused by systemic genomic instability (SGI). Additionally, we have investigated a potential mechanism that influences SGI, namely global noise in gene expression.
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    Two model systems for studying the effects of acute radiation exposure on gene deletions and amplifications
    (Colorado State University. Libraries, 2014) Sharif, Rabab S., author; Weil, Michael, advisor; Argueso, Juan Lucas, advisor; Thamm, Douglas, committee member
    Ionizing radiation (IR) poses a severe threat to genome integrity, and is an important source of environmental damage, arising from naturally occurring sources (e.g. radon and cosmic radiation) and medical imaging and therapy. Radiation exposure can lead to somatic changes in chromosomal structure such as copy number alterations (CNAs) resulting in gain or loss in copies of sections of DNA. To study copy number alterations in the human genome resulting from gamma radiation, early passage cultures of normal human fibroblasts were exposed to a single acute 4 Gy dose of radiation. Irradiated cells were kept for 48 h to allow repair of initial DNA damage. Single cell cloning was done by serial dilution in 96 well plates. Standard PCR was performed using seven sequence tagged site (STS) markers (SY 83, SY86, SY88, SY1190, SY1191, SY1201, and SY1206) of the azoospermia (AZF) region in the Y chromosome to test for microdeletions, in irradiated and non-irradiated cells. The comprehensive analysis of the molecular mechanism of copy number changes, requires a more elaborate experimental system in a model organism. Hence, we also investigated copy number alterations in diploid budding yeast cells after exposing them to two acute gamma radiation doses and detecting CNAs via a unique selection system, that involves events at two chromosomes. The copy number selective system used in our yeast samples allowed us to select for copy number alterations (duplications and deletions) in all samples after exposure to radiation, which lead to nonreciprocal translocation events formed by nonallelic homologous recombination (NAHR) mechanism. These results lead us to conclude that acute exposures to gamma radiation, induced deletions and amplifications as shown in both models. The experiments described in the thesis provide a platform for future work aimed at investigating the role low dose ionizing radiation on genome stability.