Date of Graduation

Summer 2013

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Joshua Smith

Keywords

DNA repair, nucleotide excision repair, chromatin, qPCR, Tetrahymena thermophila, Rad4

Subject Categories

Medical Molecular Biology

Abstract

DNA damage caused by ultraviolet (UV) irradiation can occur in various states of chromatin organization and is repaired mainly by nucleotide excision repair (NER). The ciliated protozoan Tetrahymena thermophila contains a transcriptionally silent germline micronucleus (MIC), where chromatin is relatively tight, and a transcriptionally active somatic macronucleus (MAC), where chromatin is relatively loose. This unique characteristic provides an opportunity to assess the effects of DNA repair at different states of chromatin organization. Genomic rearrangement occurs as portions of the MIC DNA are excised at specific internal eliminated sequences (IES) and removed prior to development of the new MAC DNA, and telomeres are added at chromosome breakage sites (CBS). The aim of this research was to investigate NER in T. thermophila by creating an assay that utilizes this genomic rearrangement to detect sites of DNA damage. An assay utilizing quantitative PCR (qPCR) and primers specific for the CBS locus Tt819 and the IES loci M- and R-Elements was developed for assessment and comparison of DNA damage and repair in the MIC and MAC following various levels of UV irradiation, and a potential locus for use in the assay is described. Additionally, a knockdown of the essential NER protein Rad4 was also developed using shRNA. Together, this assay coupled with the shRNA knockdown of Rad4 allows for further investigation of NER in T. thermophila.

Copyright

© Steven Anton Hill

Campus Only

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