Speciation is the process by which one species becomes two species that can no longer interbreed. A common hypothesis is that infertility between closely related species is due to the rapid evolution of DNA double-strand-break hotspots that are active in meiosis. DNA double-strand-breaks play an important role in meiosis by initiating recombination after chromosome replication and can be repaired as crossovers. However, hotspots where most of the breaks happen are preferentially lost to gene conversion during DNA repair. It is a mystery how hotspots are maintained over evolutionary time when they are constantly destroyed during meiosis. We hypothesize that hotspots rapidly appear and disappear over evolutionary time, which results in speciation. Schizosaccharomyces kambucha and Schizosaccharomyces pombe are two very closely related fission yeast species that are 99.5% identical at the DNA sequence level. Mapping out the hotspots of Schizosaccharomyces kambucha and comparing them to those of Schizosaccharomyces pombe will be a useful way to test our hypothesis. For this experiment, we have created Schizosaccharomyces kambucha mutants that can no longer repair the breaks so that we can pull out the broken DNA pieces using Chromatin Immunoprecipitation and locate where the DNA double-strand-break hotspots are in the genome. We expect to see the hotspots of Schizosaccharomyces kambucha differ from those of Schizosaccharomyces pombe, which will support the hypothesis that the difference in DNA double-strand-hotspots is indeed contributing to speciation. This research can be extended further to explain speciation in other organisms or unexplained infertility between human couples.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins and signatures of episodic lineage-specific positive selection suggests a function in suppressing genetic conlifcts such as segregation distortion in the male germline.

Genetic conflicts arise between two genetic elements that compete to promote their own evolutionary success. Transposons and host defense factors are a classic example. Transposons are selfish, mobile DNA elements that threaten to disrupt other genomic sequences when active. Polycomb Group (PcG) proteins, previously thought of exclusively as developmental regulators, can also function as transposon-silencers. Several PcG proteins are rapidly evolving under positive selection in Drosophila, suggesting they might be engaged in genetic conflict with transposons. My research project seeks to determine whether transposons are the biological force driving the signature of selection observed with certain PcG proteins. In order to test this, I am in the process of creating transgenic Drosophila melanogaster flies that are homozygous mutants for a given PcG gene and harbor a diverged, transgenically introduced allele of the PcG gene instead. I created the diverged PcG constructs using stitch PCR, cloning and sequencing. I plan to measure the transposon activity levels of these transgenic flies. Elevated transposon expression in flies that harbor a diverged PcG gene would suggest that transposons are responsible for the signature of selection observed at several PcG proteins.

Evolutionary conflicts involving mimicry are found throughout nature ranging in a multitude of levels from ecosystems to molecules. Moreover, structural mimicry is increasingly being recognized at the interface between viruses and hosts. The goal of my project is to provide structural and mechanistic insight into a newly discovered evolutionary ‘arms race’ between a viral protein and a host immunity protein. We have found that a poxviral protein IBP1 (IFIT Binding Protein 1) binds a host immunity protein, IFIT2, which represses viral mRNA translation. I hypothesize that IBP1 has convergently evolved to structurally mimic IFIT2’s binding partner, eIF3c, as a strategy to restore viral protein production. Because IBP1 has no known amino acid similarity to any other viral or host protein, the best way to test my hypothesis is to determine the molecular structure of IBP1 and compare it to eIF3c. I will biochemically engineer constructs to optimize the expression and amount of soluble IBP1 in E. coli and then utilize affinity columns to isolate IBP1, which will then be subjected to a myriad of crystallization conditions. Once optimized crystallization conditions have been found I will use X-ray diffraction techniques to solve the structure. Regardless of whether my hypothesis is correct, my work will provide atomic resolution detail for IBP1, which is a protein that is important for the fitness of the virus. If I find that IBP1 is indeed using molecular mimicry it will not only reveal the mechanism of IBP1 action, but it will also provide a case of molecular mimicry marked by the signature of convergent evolution. Furthermore, my work will make progress towards further understanding the interactions between hosts and viruses, which determine the results of potentially deadly infections in so many people.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States and much of the world. NAFLD resembles alcohol-induced liver injury, but occurs in patients with little to no history of alcohol consumption. It is a fairly benign condition, but can progress to a more advanced disease known as nonalcoholic steatohepatitis (NASH); many factors have been implicated in the pathogenesis of this transition (ER stress, mitochondrial dysfunction, insulin resistance, oxidative damage), but the exact mechanism remains unclear. Iron, although essential to many cellular processes, leads to increased oxidative stress and may be significant to NASH development via dysregulation of microRNA expression. To test our hypothesis, we measured microRNAs known to regulate processes marking the transition of NAFLD to NASH, such as apoptosis, inflammation, and lipid metabolism with real-time reverse-transcription PCR in a NAFLD mouse model. By comparing microRNA levels from the liver tissue of iron-treated vs. non-iron-treated NAFLD mice, we can survey effect of iron in exacerbating NAFLD via differential microRNA expression. As a regulator of cellular gene expression by transcriptional inhibition of messenger RNA, microRNA have the capacity to predict cellular processes before physiological changes are seen in the affected tissue. Thus, our results can more specifically describe differentially regulated genes during NASH development in the context of iron. After profiling our treatment groups, we will use bioinformatics to identify gene targets of the significant microRNA and validate the relationship of microRNAs and target genes in vitro. We will also compare the microRNA expression profiles of liver tissue and serum to identify serum biomarkers of NASH. In addition to the mechanistic insights elucidating the pathogenesis of NASH, our findings may lead to alternative non-invasive methods for NASH diagnosis, as liver biopsy is currently the only method for diagnosing NASH.