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Browsing Theses and Dissertations by Author "Argueso, Juan Lucas, advisor"
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Item Open Access 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 memberMultiple 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.Item Open Access 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 memberIonizing 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.