yEvo (“yeast evolution lab”) provides experimental evolution teaching modules to high school classrooms to bring genuine research experiences to students. Our goal is to show students how evolution can be applied to solve biological problems by asking them to carry out their own evolution experiments. Recently, we developed an experiment where students evolve yeast in wood hydrolysate media, which comes from lumber waste and contains plant sugars that yeast can convert to biofuel. Wood hydrolysate is a stressful condition for yeasts, as it contains acetic acid and harsh phenolic compounds, therefore yeast struggle to grow in this media. Mutations that yeast acquire while evolving in this media could be used to engineer a better biofuel-producing yeast. Students at Foster High (Tukwila, WA) evolved yeast in this media for seven weeks with transfers to fresh media every week. We retrieved the student’s evolved yeast and they showed striking new characteristics. We then used whole genome sequencing to identify underlying mutations. Some yeasts developed ring-like growths above the wood hydrolysate media and whole genome sequencing of two of these clones revealed mutations in regulators of cell-cell adhesion. Other yeasts reached very high population densities but whole genome sequencing revealed no novel mutations. I then utilized DNA staining and flow cytometry to check the DNA content of these evolved yeasts to see if changes in ploidy caused their new characteristics. The results showed that these yeast effectively became diploid, which has been shown by other labs to increase fitness in sugar-rich environments. yEvo is currently designing future experiments to further expand upon these interesting new results so that we can further understand mechanisms of adaptation to wood hydrolysate.

Despite containing over two meters of DNA, the human nucleus amazingly is still able to fit said quantity of genetic material into a space only ten micrometers in diameter. To explore this phenomenon, we first utilize computational tools to find optimal locale on strands of DNA for the deployment of synthetic oligonucleotide (oligo) hybridization probes that, when hybridized, allow us to visualize the structure of chromosomes in the nucleus using microscopy. To find these optimal regions, we use a set of programs known as OligoMiner to parse through sections of DNA, considering certain parameter restrains like melting temperature and nucleotide content ratios. One area we explore is how altering the acceptable level of the nucleobase guanine in target regions affects the number of optimal sites located on a given strand, as well as whether these findings are relevant to the reverse strand. We hope that pinpointing ratios of only guanine will allow for better hybridization efficiency, which in turn would result in increased probe effectiveness overall. To accomplish this, we first encode the aforementioned checks into OligoMiner since, in its original form, this set of programs only verifies the combined ratio of guanine and cytosine. Following the creation of these additional tools, we run OligoMiner on both random and specific sections of the human X-chromosome. We’ve seen that guanine-rich regions of DNA will see a greater concentration of probe sites on the reverse strand under our new parameter involving solely this nucleobase, compared to that of the combined cytosine ratio, due to the guanine-cytosine pairing ensuring the ratio stays constant between strand compliments. The future of this research lies both in further updating and expanding OligoMiner while also examining probe density in regions beyond just those of the X-chromosome.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is caused by the death of dopamine-secreting neurons in the midbrain. The onset of symptoms such as progressively worsening tremors, movement difficulty, and dementia are thought to be caused by protein aggregates called Lewy bodies, mitochondrial defects, and neuroinflammation. Mutations in the GBA gene, one of the genes responsible for PD symptoms, accounts for 5-10% of all PD cases. It codes for the enzyme glucocerebrosidase which breaks down the lipid glucosylceramide. Expression of GBA in muscle cells reduces protein aggregation in the head and GBA is found in extracellular vesicles (EV). Similarly, expression in the midgut partially reduces protein aggregation in the head. Our goal is to determine if GBA expressed in the midgut can travel in EVs. We have developed a GBA mutant fly model that features symptoms similar to human PD – neurodegeneration, shortened lifespan, motor deficits, and increased protein aggregation. Flies were bred and crossed to the correct genotype, collected and frozen, and processed for analysis. To ensure that GBA is not expressed elsewhere to prevent confounding, we use an Npc1b promoter with the GAL4/UAS system, since the Npc1b gene is only expressed in the midgut. Protein is quantified following gel electrophoresis and western blots. We expect to find GBA in EVs which would help confirm that GBA traveling in EVs is responsible for the reduction in protein aggregation. This is potentially an important feature for the development of treatments for PD, as understanding what causes protein aggregation is a key step in eliminating or reducing it to prevent PD.

Experimental evolution can determine genetic interactions during natural selection in complex systems. Using whole-genome sequencing of 95 parallel populations of haploid Saccharomyces cerevisiae experimentally evolved for 250 generations, we discovered a possible epistatic interaction between two sets of beneficial mutations. The first mutation is a transposable element (TE) insertion into the promoter of FLO1, giving rise to a cellular aggregation phenotype known as flocculation. The second set are putative loss of function mutations in genes encoding members of the SAGA-complex, which is thought to increase expression of genes proximal to TEs. We hypothesize that without the members of the SAGA-complex, the FLO1 gene will be unexpressed, abrogating the flocculation phenotype. We isolated three flocculant clones with TE insertions from different experimental populations and crossed them with three clones with the deletion in the SAGA-complex. Through meiosis, the yeast sporulated into four cells. The ratios of flocculant to wildtype haploid cells are used to determine an epistatic interaction. A 2:2 ratio suggests a non-epistatic interaction while a 1:3 flocculant to wildtype ratio suggests an epistatic interaction. The project is in the early stages but segregation ratios suggest the members of the SAGA-complex with deletion mutations do not hinder the expression of the FLO1 gene. Our alternate hypothesis is members of the SAGA-complex have no effect on the activation of TE insertions that promote the expression of the FLO1 gene. While the initial hypothesis might not hold, this experiment will give us a further understanding of genetic interactions during evolution.

Prostate cancer (PCa) is the most commonly diagnosed and second most deadly cancer in men. Almost all of these deaths are the result of a very progressed form called metastatic, castration-resistant prostate cancer (mCRPC), which currently has no cure and is incompletely understood. Cancer-related mutations in the untranslated regions (UTRs) of mRNA transcripts have been found to contain various sequence or structural motifs that contribute to the regulation of these cancer-causing genes. These regions are extremely dynamic in their control over gene expression affecting mRNA stability and translation efficiency which have both been previously implicated in prostate cancer. However, the degree to which these mutations in the UTRs functionally contribute to prostate cancer remains poorly understood – especially in the 3’ untranslated region (3’UTR). A candidate gene list to investigate was constructed from an analysis of patient tumor sequencing data from a broad cohort of 230 localized and metastatic prostate cancer patients. I Gibson cloned wild type (WT) and mutant 3’UTRs from the candidate genes into luciferase plasmid constructs. Subsequent dual luciferase assay data revealed significant changes in protein expression between WT and mutant constructs most notably in the genes NCL and CLEC18B. Nucleolin (NCL) is a protein involved in the synthesis and maturation of ribosomes and is oncogenic in many cancers when overexpressed, while CLEC18B is largely unstudied. Given this existing functional evidence and my preliminary data, further investigation into the differential expression of NCL and the cellular mechanism through which it is achieved is warranted. My project focuses on elucidating the effects of 3’UTR somatic mutations on translational regulatory regions of the human genome, so that we may uncover new patterns in the progression of prostate cancer and subsequently elicit possible novel therapeutic targets with which to better treat these patients.