USING SOUTHERN BLOTTING AND NON-RADIOACTIVE PROBE HYBRIDIZATION AS A TOOL TO MEASURE 2’,3’-DIDEOXYCYTIDINE INDUCED MITOCHONDRIAL DNA DEPLETION IN HUMAN CELL LINES

Author

Joel Wheeler, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2019

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Young, Matthew

Abstract

Mitochondria are membrane bound organelles important for energy production in respiring cells through the process of oxidative phosphorylation. They have their own multi-copied mitochondrial DNA (mtDNA) genome separate from that in the nucleus that is needed for mitochondria to function properly and can exist in both wild type and mutant forms in the same cell. The integrity of the mtDNA is therefore of vital importance for the survival of the organism and as such understanding the mechanisms of mtDNA maintenance is relevant to human health and disease. This study employs a Southern blotting and non-radioactive probe method to examine various aspects of mtDNA maintenance. Restriction endonuclease mapping utilizing mtDNA-specific and nuclear DNA-specific digoxigenin (DIG)-labeled probes was performed to show that the synthesized probes are indeed specific for their target sequences. The DIG-labeled probes were used to quantitate mtDNA content from different DNA isolation methods. Whole-cell DNA extraction was found to yield higher levels of mtDNA compared to a commercially available spin-column kit. Next, Southern blots were used to analyze mtDNA copy number as well as mtDNA depletion in the hepatocarcinoma-derived cell line HepaRG following exposure to the nucleoside reverse transcriptase inhibitor 2’,3’-dideoxycytidine (ddC), a known mitochondrial toxicant. In comparison to proliferative HepaRG differentiated HepaRG contained about 2-fold more mtDNA. Relative to untreated control cells, proliferating HepaRG exposed to ddC had greater than a 96% reduction in mtDNA and had decreased cellular viability. Differentiated HepaRG cell viability was not affected after 13 days of ddC treatment; however, significant mtDNA depletion was observed. We estimate that differentiated HepaRG mtDNA depletion occurs quickly at about 20 molecules per hour.