creator Beatty, Evan Alexander 2019 Spring 2019 Thesis Illinois Institute of Technology BIOL / Biology advisor Xiang, Jialing Molecular biology Biochemistry Biology BAXΔ2 Biosynthesis Dual Luciferase Reporter Assay Programmed Ribosomal Frameshift Reading Frame Shift Recoding Events en 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. born digital application/pdf In Copyright http://rightsstatements.org/page/InC/1.0/ Restricted Access http://hdl.handle.net/10560/islandora:1000805