ATTENTION: The software behind KU ScholarWorks is being upgraded to a new version. Starting July 15th, users will not be able to log in to the system, add items, nor make any changes until the new version is in place at the end of July. Searching for articles and opening files will continue to work while the system is being updated.
If you have any questions, please contact Marianne Reed at mreed@ku.edu .
The effect of copy number variation at the Cyp12d1 gene on caffeine toxicity in Drosophila melanogaster
dc.contributor.advisor | Macdonald, Stuart J | |
dc.contributor.author | Hernandez-Aquino, Dianarys | |
dc.date.accessioned | 2024-01-25T21:38:49Z | |
dc.date.available | 2024-01-25T21:38:49Z | |
dc.date.issued | 2020-12-31 | |
dc.date.submitted | 2020 | |
dc.identifier.other | http://dissertations.umi.com/ku:17562 | |
dc.identifier.uri | https://hdl.handle.net/1808/34918 | |
dc.description.abstract | 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. | |
dc.format.extent | 46 pages | |
dc.language.iso | en | |
dc.publisher | University of Kansas | |
dc.rights | Copyright held by the author. | |
dc.subject | Genetics | |
dc.subject | Molecular biology | |
dc.subject | Caffeine Resistance | |
dc.subject | CNV | |
dc.subject | Cyp12d1 | |
dc.title | The effect of copy number variation at the Cyp12d1 gene on caffeine toxicity in Drosophila melanogaster | |
dc.type | Thesis | |
dc.contributor.cmtemember | Blumenstiel, Justin P | |
dc.contributor.cmtemember | Unckless, Robert L | |
dc.thesis.degreeDiscipline | Molecular Biosciences | |
dc.thesis.degreeLevel | M.S. | |
dc.identifier.orcid |
Files in this item
This item appears in the following Collection(s)
-
Theses [4088]