Azole drug resistance in fungi is a well-established phenomenon. Previous research through the yEvo (yeast Evolution) program used Saccharomyces cerevisiae as a model organism to study how azole resistance arises using experimental evolution. By growing yeast in increasing doses of azole over time, high school students selected for yeast cells that gained favorable mutations for azole resistance. Sequencing this yeast at UW enabled us to identify specific mutations that contribute to azole resistance. We collaborated with Fred Hutch Science Education Partnership to design a lesson kit that can be checked out by local high school instructors for use in their classrooms. We selected twelve strains from our previous azole evolution experiments that contained a variety of mutations. These included missense and synonymous mutations, copy number gains, transposon insertions, and mitochondrial DNA loss. To develop this kit, I tested the experimental conditions by growing individual strains in a range of azole concentrations. From this, I chose an azole concentration that sufficiently introduces environmental pressure but still allows for strain growth. I then performed a series of competitive growth experiments to confirm replicability and test the procedure as it would be used in the kit. Using the results of my tests, I also contributed to creating the accompanying protocol and curriculum for the kit. Students will have the opportunity to make predictions through a bracket-style match-up, learning about each strain through “trading cards” that I am helping design to contain information about the mutations of each strain. In the final step of this project, I will take part in a training session to support high school instructors interested in teaching the kit. From the implementation of this kit, students will learn broadly about the effects of different types of mutations, and specifically how mutations affect anti-fungal drug resistance.

Uridine-5'-triphosphate (UTP) is a precursor for RNA synthesis. Ura3 catalyzes the conversion of orotidine-5'-phosphate (OMP) into uridine monophosphate (UMP) and is commonly used as a selection marker to characterize mutation rates of S. cerevisiae. URA3 can be positively selected for by growing cells in the absence of uracil and can be selected against by growing cells in the presence of the toxic fluorinated UTP precursor, 5-Fluoroorotic acid (5-FOA). While mutations in URA3 make up the majority of 5-FOA-resistant mutants, mutations in a small number of other loci can also cause this phenotype. We whole genome sequenced the 5-FOA-resistant mutants with a wild type URA3 and identified mutations to URA6 in each of these individuals. URA6 is an essential gene that encodes an enzyme that catalyzes the conversion of uridine monophosphate (UMP) into uridine-5'-diphosphate (UDP). Here, we describe 41 non-synonymous mutations to URA6 that permit growth in both the absence of uracil and in the presence of 5-FOA. It remains unclear how the URA6 mutants can maintain a functioning UTP synthesis pathway while remaining resistant to the toxic fluorinated precursors. We hypothesize that these mutations alter the protein structure in a manner that decreases the affinity for fluorinated substrates while maintaining the affinity for UDP. To test this, we will evaluate the structural changes to URA6 resulting from these non-synonymous mutations. Our goal is that our findings can benefit our understanding of the UTP biosynthesis pathway and encourage further investigation of the mechanisms involving fluorinated substrate analogues.

Saccharomyces cerevisiae is a model organism that is essential in the production of products such as wine, bread, beer, and bioethanol. The process of domestication through selection of desired traits for beer brewing has led to genomic changes in these S. cerevisiae strains. Specifically, the brewing process creates conditions that favor asexual reproduction as opposed to sexual reproduction, allowing genomic changes detrimental to meiosis to accumulate. Some genomic changes resulting from domestication include aneuploidy, genome decay, and high copy number variation. Decreased ability to undergo meiosis makes genetic linkage studies like quantitative trait loci (QTL) mapping incredibly difficult compared to lab strains. Meiosis is a key part of QTL mapping, where a parental strain for a phenotype of interest undergoes meiosis to generate progeny with variation in the phenotypic trait and in their genotypes. My work aims to find and develop genetically tractable brewing yeast strains in order to perform QTL mapping on unique brewing traits. The brewing trait of interest to my work is thermotolerance, as higher temperatures around the globe result in harsher selection conditions on brewing yeast. Previous work on Norwegian kveik strains revealed high thermotolerance and the ability to undergo meiosis and produce viable offspring. My project aims to understand the genetic basis of increased thermotolerance in kveik strains. I will conduct heat tolerance assays to determine the effect a select range of temperatures has on growth. I expect to see variation between individuals in a population and the variation will allow me to conduct bulk segregant analysis–the specific type of QTL mapping I aim to do–for the genotypes associated with the increased thermotolerance trait. As global temperatures are rising more rapidly, it is essential to understand how organisms use thermotolerance as an adaptive response.