Two model systems for studying the effects of acute radiation exposure on gene deletions and amplifications

Abstract

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.