Detection Of BAXΔ2 Reading Frame Shift Using A Dual Luciferase Reporter System Beatty, Evan Alexander Molecular biology Biochemistry Biology BAXΔ2 Biosynthesis Dual Luciferase Reporter Assay Programmed Ribosomal Frameshift Reading Frame Shift Recoding Events While initial studies of the pro-apoptotic Bcl-2-associated X protein isoform Δ2 (BAXΔ2) identified the combination of an alternative splicing event and a gene-level mutation as the prerequisites for biosynthesis in microsatellite unstable (MSI+) human colon cancer cells, no similar explanation existed to explain the presence of this protein in normal and normal adjacent tissues. To identify an alternative to the gene-level mutation in the absence of an MSI+ phenotype, we utilized a dual luciferase reporter assay designed to observe epigenetic recoding. Plasmid constructs containing the first two exons encoding BAXΔ2 were either transcribed and translated in vitro or transfected into BAX-negative human colon cancer cells. In both cases, assay of the protein products of the reporter genes demonstrate that a low level (2.82% in vitro, 4.43% in vivo) of all translational events which produce the protein product of an upstream reporter gene also produce the protein product of a downstream reporter gene. This occurs despite the two existing in different reading frames as a result of the BAX exons cloned between them. These results confirm that an epigenetic recoding event is able to salvage the BAX reading frame in cases where exon 2 has been excised, and further narrow down the potential mechanism involved to either transcriptional slippage or programmed ribosomal frameshifting. Xiang, Jialing 2019 Thesis application/pdf islandora:1000805 http://hdl.handle.net/10560/islandora:1000805 Illinois Institute of Technology BIOL / Biology en In Copyright http://rightsstatements.org/page/InC/1.0/ Restricted Access