Dysfunctional Gene Regulation of the Meiotic Program in an Interspecific Yeast Hybrid

Speciation, the evolutionary process by which species arise, is a fundamental biological concept. One of the major goals of evolutionary genetics is to understand the genetic basis of reproductive isolation (RI), a collection of barriers that prevents two species from forming viable or fertile hybrids. Given the lack of viable or fertile hybrid progeny, identifying genes that impact RI as well as their functions has been difficult. Thus we lack a view of the total genetic contribution to RI. Recently the budding yeast Saccharomyces has served as an evolutionary model, as abundant sequence, expression, and phenotypic data exist for the model organism S. cerevisiae and its closest relatives. Additionally we can manipulate the yeast genome and control its environment arguably more than any other organism. Hence I developed assays to catalog all genes contributing to RI between S. cerevisiae and its closest known relative S. paradoxus, which can form sterile hybrids under laboratory conditions. Chapter 2 details my utilization of accessible genetic tools for yeast to understand the total contribution of genes to RI. Though I unveiled multiple problems with studying speciation genetics using standard methods in yeast, I acquired valuable information about the biology of hybrids. For instance, I determine that yeast hybrids are highly sensitive to background mutations, commonly generated in yeast transgenesis, resulting in experimental artifacts. Using this knowledge, I took advantage of the emergence of next-generation sequencing in Chapter 3 to analyze wild type hybrid and parental genome expression to understand the relationship between gene expression and RI. My main objectives in my dissertation are to understand dysfunctional hybrid gene regulation in the context of RI in yeast and to ascertain subsets of genes whose expression is disrupted. Thus I measured genome-wide changes in gene expression over the course of meiosis for S. cerevisiae, S. paradoxus and their sterile hybrid. I show that misexpressed genes in a yeast hybrid result from earlier activation of the meiotic program relative to its parents. This heterochrony is expected under the anti-recombination model of RI in yeast. I also find an increase in dysfunctional regulation in genes that are involved with sporulation, mitochondrial function, rRNA processing and translation. Genes in these pathways could contribute to RI. My dissertation adds to the field of speciation genetics, as it lends an example of a time-dependent relationship between dysfunctional hybrid regulation and RI for yeast species, as well as identifies candidate genes that could contribute to RI

A Catalog of Neutral and Deleterious Polymorphism in Yeast

The abundance and identity of functional variation segregating in natural populations is paramount to dissecting the molecular basis of quantitative traits as well as human genetic diseases. Genome sequencing of multiple organisms of the same species provides an efficient means of cataloging rearrangements, insertion, or deletion polymorphisms (InDels) and single-nucleotide polymorphisms (SNPs). While inbreeding depression and heterosis imply that a substantial amount of polymorphism is deleterious, distinguishing deleterious from neutral polymorphism remains a significant challenge. To identify deleterious and neutral DNA sequence variation within Saccharomyces cerevisiae, we sequenced the genome of a vineyard and oak tree strain and compared them to a reference genome. Among these three strains, 6% of the genome is variable, mostly attributable to variation in genome content that results from large InDels. Out of the 88,000 polymorphisms identified, 93% are SNPs and a small but significant fraction can be attributed to recent interspecific introgression and ectopic gene conversion. In comparison to the reference genome, there is substantial evidence for functional variation in gene content and structure that results from large InDels, frame-shifts, and polymorphic start and stop codons. Comparison of polymorphism to divergence reveals scant evidence for positive selection but an abundance of evidence for deleterious SNPs. We estimate that 12% of coding and 7% of noncoding SNPs are deleterious. Based on divergence among 11 yeast species, we identified 1,666 nonsynonymous SNPs that disrupt conserved amino acids and 1,863 noncoding SNPs that disrupt conserved noncoding motifs. The deleterious coding SNPs include those known to affect quantitative traits, and a subset of the deleterious noncoding SNPs occurs in the promoters of genes that show allele-specific expression, implying that some cis-regulatory SNPs are deleterious. Our results show that the genome sequences of both closely and distantly related species provide a means of identifying deleterious polymorphisms that disrupt functionally conserved coding and noncoding sequences

Evaluation to Improve a High School Summer Science Outreach Program

The goal of the Young Scientist Program (YSP) at Washington University School of Medicine in St. Louis (WUSM) is to broaden science literacy and recruit talent for the scientific future. In particular, YSP seeks to expose underrepresented minority high school students from St. Louis public schools (SLPS) to a wide variety of careers in the sciences. The centerpiece of YSP, the Summer Focus Program (SFP), is a nine-week, intensive research experience for competitively chosen rising high school seniors (Scholars). Scholars are paired with volunteer graduate student, medical student, or postdoctoral fellow mentors who are active members of the practicing scientific community and serve as guides and exemplars of scientific careers. The SFP seeks to increase the number of underrepresented minority students pursuing STEM undergraduate degrees by making the Scholars more comfortable with science and science literacy. The data presented here provide results of the objective, quick, and simple methods developed by YSP to assess the efficacy of the SFP from 2006 to 2013. We demonstrate that the SFP successfully used formative evaluation to continuously improve the various activities within the SFP over the course of several years and in turn enhance student experiences within the SFP. Additionally we show that the SFP effectively broadened confidence in science literacy among participating high school students and successfully graduated a high percentage of students who went on to pursue science, technology, engineering, and mathematics (STEM) majors at the undergraduate level