Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor

Kuenzel, Wayne

Reader

Allison, Neil

Second Reader

Hagstrom, Fran

Third Reader

Kumar, Suresh

Abstract

Chronic stress is a problem not only in terms of its psychological effects but also due to the harmful physiological changes induced in the individual. Although chronic stress is recognized as detrimental and rampant throughout society, the stress system itself remains poorly understood in terms of comprehending which genes are involved in regulating and carrying out stress-induced signals. In a previous study, 57 candidate genes in the stress pathway had been identified based on the presence of stress-associated single nucleotide polymorphisms (SNPs) and differential expression from microarray data. The objective of this study was to investigate Additional Sex Combs-Like 2 (ASXL2), one of those 57 candidate genes, and to determine if it was part of the stress pathway through SNP identification and Real Time-Polymerase Chain Reaction (RT-PCR). Utilizing sequencing data of ASXL2 from Pubmed as well as SNP locations generated from DNASTAR’s SeqMan Ngen Assembly, SNPs were identified within the actual reference genome verifying the normal and mutant base pair for each SNP. After identifying SNPs of ASXL2 within the low stress line of Japanese quail, RT-PCR was performed in order to determine if mRNA expression for ASXL2 would change under acute and chronic stress. In order to perform RT-PCR, primers were designed and then tested utilizing gel electrophoresis in order to ensure optimization of the PCR product. ΔΔCT methodology was then used to determine the veracity of the mean fold expression changes found for ASXL2 under acute and chronic stress. As part of the experiment to determine the potential role of ASXL2 in the stress pathway, four SNPs were identified with high SNP rates in the low stress line of Japanese quail within ASXL2. After performing RT-PCR, it was found that ASXL2 was up regulated by 14% under acute stress and down regulated by 24% under chronic stress in comparison to the acute control group and chronic control group respectively. P-values of 0.0013 and 0.0004 for acute and chronic stress respectively indicate that the data are statistically significant. β-Actin was set at a value of 1.0 as an internal control in order to determine mean fold expression changes for ASXL2 under differing stress conditions by using ΔΔCT methodology. Based on the high SNP rate as well as the differential expression of ASXL2 under acute and chronic stress, ASXL2 has been directly implicated as a stress-related gene. Although ASXL2’s exact role within the stress pathway is unknown, its interactions with trithorax and Polycomb group genes as an epigenetic modifier make it an interesting candidate for further research to determine its possible role in the epigenetic effects of acute and chronic stress.

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