The effect of copy number variation at the Cyp12d1 gene on caffeine toxicity in Drosophila melanogaster
Issue Date
2020-12-31Author
Hernandez-Aquino, Dianarys
Publisher
University of Kansas
Format
46 pages
Type
Thesis
Degree Level
M.S.
Discipline
Molecular Biosciences
Rights
Copyright held by the author.
Metadata
Show full item recordAbstract
Xenobiotic compounds are a constant challenge for animals. We desire to understand the mechanisms behind xenobiotic resistance by identifying loci underlying variation in the toxicity of such compounds. Previous work in D. melanogaster mapped several loci contributing to resistance to caffeine, a model xenobiotic compound, one of which implicated the cytochrome P450 gene Cyp12d1. Both through RNAi knockdown and RNAseq, Cyp12d1 involvement in caffeine resistance was detected, moreover, an association between greater Cyp12d1 copy number and increased caffeine resistance was seen. To further test this, we used CRISPR-Cas9 editing to generate putative loss-of-function mutations in exon 2 of Cyp12d1, confirming via RNAseq that mutation-carrying strains have significantly lower Cyp12d1 expression than our single control strain. We subsequently measured caffeine resistance in all strains, observing a significant reduction in resistance in 2 out of 3 CRISPR mutant strains relative to the control strain. Interestingly, the third mutant strain exhibited higher resistance to caffeine than the control. We present evidence suggesting that this result is due to a difference in splice site mutations. This makes it challenging to compare directly among strains. To overcome this, and to explicitly enable a test of the effect of varying Cyp12d1 copy number, we next edited a strain containing two, nearly identical copies of Cyp12d1. Creating a strain with one perfect copy of Cyp12d1 while preserving the genetic background of the original genotype, as well as a control non-mutated strain with two copies of Cyp12d1. We observed a trend, but no significant difference in resistance was detected between the lines. Using these CRISPR lines we then performed RNAseq on guts of female flies to measure RNA levels under naïve and caffeinated conditions. To which, we were able to see a significant increase in Cyp12d1 RNA levels correlated to an increase in gene copy number. All together, we confirmed the involvement of Cyp12d1 in caffeine resistance, but more work is needed to determine the role of copy number variation at Cyp12d1.
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