Using Fluorescence Assays to Explore the Regulation of the Kynurenine Pathway in Neurospora crassa
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Date
2021
Authors
Speed, Haley
Journal Title
Journal ISSN
Volume Title
Publisher
University of Oregon
Abstract
In Neurospora crassa (a filamentous fungus), there are several enzymes that cause the breakdown of tryptophan into fluorescent anthranilic acid, several of whose genes have the chromatin markers that our lab studies, specifically methylation of lysine 27 of histone H3. If we give a N. crassa culture tryptophan and it fluoresces, this indicates that the genes are “turned on” normally; if it doesn't, they could be abnormally “turned off.” Since July of 2020, I have been using this convenient system to gain insights into the mechanism controlling tryptophan degradation, which may illuminate general chromatin control processes. Utilizing the FGSC knockout library, I have screened 13,000 mutants using fluorescence assays, leading me to identify eleven that fluoresce atypically. I used 150 μL liquid cultures for my primary screening and 1 mL liquid cultures for my secondary screening of the collection. I have also confirmed the knockouts via PCR and am currently complementing the genes of interest to determine if the knockouts are causing the phenotype. The next steps of this research is to find out why my mutants are behaving abnormally, and to determine if this is because of abnormalities in their chromatin markers. Studying chromatin markers is essential to understanding the eukaryotic genome at large because of their ubiquity throughout most eukaryotic organisms.
Description
1 page.
Keywords
H3K27me, Tryptophan, Kynurenine Pathway, Neurospora crassa