<p> The vacuole in <i>Saccharomyces cerevisiae</i> serves as a model for the mammalian lysosome. In a genome wide screen for mutants with severe growth <u>h</u>ypersensitivity to <u>h</u>ygromycin B, our lab identified 14 <i>HHY</i> genes. Each of the <i> hhy</i> mutants is defective in vacuolar trafficking and/or function and also sensitive to rapamycin and caffeine, suggesting a compromised target of rapamycin (TOR) kinase pathway. My research divides the <i>hhy</i> mutants into two groups based on quantitative growth analyses in the presence of hygromycin B. (1) a super affected group (<i>s-hhy's</i>) and (2) a dose-dependent group (<i>d-hhy's</i>). The <i>s-HHY</i> genes include <i>CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52,</i> and <i>VPS54</i>. Evaluation of the known functions of <i> s-HHY</i> gene products reveals vesicular trafficking function at the trans-Golgi and late endosome interface to be a common factor. In yeast, the TORC1 complex localizes to the vacuole. Because a compromised TORC1 complex signaling is suggested in <i>hhy</i> mutant strains due to their caffeine or rapamycin sensitivity, I hypothesized that compromised TORC1 signaling in <i>HHY's</i> may be due to defects in the vacuolar localization of Tor1 kinase. To assess Tor1 kinase localization, we utilized a strain expressing endogenously tagged Tor1-GFP and assayed localization to the vacuolar membrane in each of the s-hhy mutants using confocal microscopy. In wild-type cells, Tor1-GFP co-localizes with the vacuolar membrane marker FM4-64 while s-hhy deletion strains fail to localize Tor1-GFP to the vacuolar membrane when treated with hygromycin B. Our results implicate that Tor1p is transported to the vacuole membrane via the late endosome (CPY pathway) and not the ALP (Vam3) pathway. Additionally, <i>s-hhy</i> mutants are unable to recover growth after a 4-hour treatment with hygromycin B, similar to EG0 mutants, which fail to exit from G0 after treatment with the TORC1 inhibiting drug rapamycin. Based on our data, we propose a model in which the <i>s-HHY </i> gene functions in vesicular trafficking at the trans-Golgi/late endosome interface are involved in recruitment and subsequent transport of Tor1p to the vacuolar membrane, and that interface is hypersensitive to hygromycin B. We also propose that Tor1 kinase localization at the vacuole is essential for its cell cycle regulatory function.</p>

<p>The success of Drosophila as a system for genetic analysis is closely linked to its amenability to genetic manipulation. Part 1 of the dissertation elucidates a novel scheme for long-range targeted manipulation of genes. We integrated an 80-kb genomic fragment at its endogenous locus, utilizing a targeted attP attachment site for the phiC31 integrase. We achieved single-copy reduction of the resulting region duplication by inducing recombinational DNA repair. We showed that this two-step scheme of integration and reduction is efficient and useful for delivering modifications. We established a vector configuration that facilitates the recovery of modifications. The integrating genomic fragment allowed for delivery of a new attachment site at 70 kb from the existing <i>attP</i> into a new locus, making it susceptible to targeted mutagenesis. We extrapolate that with this scheme, only 1 200 lines bearing att-sites throughout the genome would suffice to render all Drosophila genes amenable to targeted mutagenesis. Excitingly, this method should be readily applicable to other systems. </p><p> In Part 2 of the dissertation, I explored the question of telomere organization in Drosophila. Telomeres demarcate the ends of linear chromosomes to distinguish them from broken ends. In yeast, they cluster at the periphery of the nucleus establishing a compartment of silent chromatin. To bring insight into telomere organization in a higher organism, we followed EGFPlabeled Drosophila telomeric protein HOAP <i>in vivo</i> and found that the 16 telomeres cluster into 4-6 foci per nucleus in somatic tissues. Interestingly, HOAP signal intensity in the clusters doubles in interphase, potentially due to loading of HOAP to newly replicated telomeres. We tested several predictions about rules governing clustering. First, by inspecting mutant embryos that develop as haploids, we found that clustering is not mediated by associations between homologs. Second, by demonstrating clustering capability for a telomere of novel sequence, we eliminated DNA sequence homology and identity as important factors. Third, by marking both ends of a chromosome, we ruled out predominance of intra-chromosomal interactions. We propose that clustering is indiscriminate of sequence and is likely maintained by a yet undetermined factor. </p>

<p> The formation of mineralized tissue was critical to the evolution and diversification of metazoans and remains functionally significant in most animal lineages. Of special importance is the protein found occluded within the mineral matrix, which facilitates the process of biomineralization and modulates the final mineral structure. These skeletal matrix proteins have well been described in several species, including the sea urchin <i> Stronglyocentrotus purpuratus,</i> an important model organism. Biomineralization research is limited in other echinoderm classes. This research encompasses the first description of mineral matrix proteins in a member of the echinoderm class Ophiuroidea. This work describes the skeletal matrix proteins of the brittle star <i>Ophiocoma wendtii</i> using bioinformatic and proteomic techniques. General characteristics of matrix protein are described and a number of candidate biomineralization related genes have been identified, cloned, and sequenced. The unique evolutionary and biochemical properties of brittle star skeletal matrix proteins are also described.</p>

<p> Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study. </p><p> The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using <i>in vitro</i> splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures. </p><p> Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa). </p><p> Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.</p>

<p> Viruses have long been noted to be composed simply of nucleic acid and protein. This thesis describes this confluence of science of viruses at the interface of genomics and proteomics. Chapter 2 describes the discovery of klassevirus, a new picornavirus in pediatric diarrhea. Chapter 3 shows that klassevirus is likely a human pathogen given the seroconversion of klassevirus-positive individuals against a klassevirus non-structural protein that is not present in the picornavirus virion. Subsequent work failed to obtain a culturable virus from klassevirus-positive stool samples, enabling the transition to culture-independent methods of characterizing picornavirus-host protein interactions. Chapter 4 describes the use of affinity purification mass spectrometry to discovery a novel picornavirus 3A-ACBD3-PI4KB complex that promotes viral replication in the enteroviruses and kobuviruses. Chapter 5 extends upon the methodology to describe a novel host protein interactor of ACBD3 (TBC1D22A/B), whose interaction is altered specifically by the kobuvirus 3A protein. This complex also demonstrates significant interaction with the klassevirus 3A protein, suggesting that the AP-MS work may inform the biology of the uncultured virus. Finally, chapter 6 describes future directions that are opened up by this work.</p>

<p> While it is interesting to examine the evolutionary history and phylogenetic relationship between species, for example, in a sort of "tree of life", there is also a great deal to be learned from examining population structure and relationships within species. A careful description of phylogenetic relationships within species provides insights into causes of phenotypic variation, including disease susceptibility. The better we are able to understand the patterns of genotypic variation within species, the better these populations may be used as models to identify causative variants and possible therapies, for example through targeted genome-wide association studies (GWAS). My thesis describes a model of local phylogenetic structure, how it can be effectively derived under various circumstances, and useful applications and visualizations of this model to aid genetic studies. </p><p> I introduce a method for discovering phylogenetic structure among individuals of a population by partitioning the genome into a minimal set of intervals within which there is no evidence of recombination. I describe two extensions of this basic method. The first allows it to be applied to heterozygous, in addition to homozygous, genotypes and the second makes it more robust to errors in the source genotypes. </p><p> I demonstrate the predictive power of my local phylogeny model using a novel method for genome-wide genotype imputation. This imputation method achieves very high accuracy&mdash;on the order of the accuracy rate in the sequencing technology&mdash;by imputing genotypes in regions of shared inheritance based on my local phylogenies. </p><p> Comparative genomic analysis within species can be greatly aided by appropriate visualization and analysis tools. I developed a framework for web-based visualization and analysis of multiple individuals within a species, with my model of local phylogeny providing the underlying structure. I will describe the utility of these tools and the applications for which they have found widespread use.</p>

<p> Identity and intensity of parasitism have been shown to be correlated to the host genotype at the major histocompatibility complex (MHC), a genomic region involved in the adaptive immune response. However, the evolutionary mechanisms by which parasites exert a selective force on host immune systems are unknown. This thesis investigates the relationship between parasitism and MHC genotype in two bear populations in Southcentral Alaska. We diagnosed infection using the polymerase chain reaction (PCR), targeting the ribosomal subunit of 18S of common blood parasites. Parasitism was detected in half of brown bears and 75% of black bears. We detected <i>Eimeria</i> spp. and several species of apicomplexan and nematode parasites, and we report here the first finding of <i>Trypanosoma cruzi</i> in Alaskan bears. We found no association between MHC genotype and identity or intensity of infection, suggesting that other loci or even non-genetic factors are important covariates in predicting infection status.</p>

<p>In skeletal muscle, loss of neuronal nitric oxide synthase (nNOS) from the sarcolemma has been observed in a few muscular dystrophies and myopathies. However, the extent of this phenomenon, its mechanism, and its physiological impact are not well understood. Using immunofluorescent staining for nNOS, a survey of 161 patient biopsies found absent or reduced sarcolemmal nNOS in 43% of patients. Patient mobility and muscle functional status correlated with nNOS mislocalization from the sarcolemma. Mouse models of inherited and acquired myopathies showed similar loss of sarcolemmal nNOS and impaired mobility and muscle function. A proteomic approach, using mass spectrometry and differentially labeled control and steroid-induced myopathy (SIM) mouse samples, found novel nNOS binding proteins including alpha-actinin-3 (ACTN3), which exhibited decreased interaction with nNOS after steroid treatment. It revealed a potential explanation for impaired muscle function in SIM as nNOS interactions were lost at the sarcomere and gained at the sarcoplasmic reticulum impairing contractility. Treating nNOS-deficient mice with steroids demonstrated that loss of sarcolemmal nNOS reduces muscle contractility and strength in SIM through increased nitric oxide (NO) signaling. In SIM mice treated with a nitric oxide donor and steroids, nitric oxide partially protects the muscle from atrophy and improves muscle fatigability and recovery suggesting nNOS mislocalization also decreases NO availability. These findings show that loss of sarcolemmal nNOS is a common phenomenon that negatively impacts muscle function. Therapeutic strategies targeting nNOS or NO signaling need to allow for the complexity of local nitric oxide content and cellular context. </p>

<p>Centromeres are the chromosomal loci where microtubule spindles bind, via the kinetochore, during mitosis and meiosis. Paradoxically the centromere, as a functional unit, is essential to guarantee faithful chromosome segregation, whereas its underlying DNA sequences and associated kinetochore proteins are fast evolving. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. In spite of their importance, very little is known about the degree to which centromeric tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from species using publicly available genomic sequence and our own data. We found that despite an overall lack of sequence conservation, centromeric tandem repeats from diverse species showed similar modes of evolution. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. In addition, we performed a survey of fungi genomes for the presence of high-copy tandem repeats, but found little evidence to suggest that high-copy centromeric repeats are a common feature feature in fungi, with the possible exception of the <i>Zygomycota</i>. phylum. Finally, in most species the kinetochore assembles at a single locus, but in some cases the kinetochore forms along the entire length of the chromosomes forming holocentric chromosomes. Following a literature review we estimate that holocentricity is very common and has evolved at least thirteen times.

<p> In this work, I describe my doctoral work studying the regulation of transcription with both computational and experimental methods on the natural genetic variation in a population. This works integrates an investigation of the consequences of polymorphisms at three stages of gene regulation in the developing fly embryo: the diversity at <i>cis</i>-regulatory modules, the integration of transcription factor binding into changes in chromatin state and the effects of these inputs on the final phenotype of embryonic gene expression.</p>