Eukaryotic DNA replication is tightly regulated during the initiation phase to ensure that the genome is copied only once and at the proper time during each cell cycle. During replication initiation, over twenty different proteins are recruited to each origin of replication to denature the DNA duplex and assemble a functional replication fork. Of these, Mcm10 is a DNA binding protein that is recruited to origins in early S-phase and is required for the activation of Mcm2-7, the replicative DNA helicase. Importantly, Mcm10 is necessary for subsequent loading of downstream replication proteins, including DNA polymerase α-primase (pol α), onto chromatin. Mcm10 interacts with single- and double-stranded DNA, pol α, as well as other proteins involved in DNA synthesis. Despite its importance in both replication fork assembly and progression, the precise role of Mcm10 remains undefined. In order to better understand the importance of the molecular interactions of vertebrate Mcm10, we have carried out a structure-function study of the protein from Xenopus laevis (XMcm10), which shares a high sequence homology with the human ortholog. XMcm10 contains three structured regions: a putative oligomerization domain at the N-terminus (NTD) and two independent DNA and pol α binding regions located in the internal (ID) and C-terminal (CTD) domains of the protein. We present a biochemical characterization of the individual domains in Chapter 2, followed by the three-dimensional solution NMR structure and DNA binding activity of the CTD in Chapter 3. The results reveal how the CTD zinc cluster binds DNA and suggests a putative role for this motif in protein-protein interactions with other replisome components. In addition, we show using XMcm10 constructs spanning the two DNA binding domains that the region between is flexible in solution, and that this linker is necessary for optimal DNA binding by XMcm10. Finally, preliminary structural evidence for how the individual ID and CTD modules coordinate DNA binding in the context of the full-length protein is presented in Chapter 4. This modular DNA binding strategy is discussed in terms of Mcm10s role in during replication initiation and elongation.

The genome is a dynamic system storing the genetic information responsible for the astounding diversity of life and examination of its variability is crucial to understanding the phenotypic differences between and within species. In this dissertation, I combined functional, population genetic and evolutionary genomic experiments to study three major facets of genome evolution and function in the filamentous fungal genus Aspergillus, a group with far reaching effects on human society: (1) the pathogenicity of A. fumigatus, (2) the domestication of A. oryzae and (3) the function and evolution of tandemly repeated DNA. The first portion centers on the in vitro examination of the transcriptional profile of A. fumigatus during its infective biofilm like state. This examination revealed widespread up-regulation of genes likely contributing to virulence (e.g. toxin-encoding gene clusters), drug resistance (e.g. transporters) and biofilm morphology (e.g. hydrophobins). Additionally, this facet includes a population genetics study of drug resistant and susceptible isolates of A. fumigatus from the Netherlands, where drug resistant isolates first originated. This study provided evidence of varying recombination rates between populations as well as the pattern of dissemination of antifungal resistance. The second theme also includes an examination of the genomic features differentiating A. flavus from its domesticated ecotype A. oryzae used in the production of traditional Japanese foods and beverages such as sake and soy sauce. Examination of the A. oryzae transcriptome and proteome showed several changes likely involved in domestication including reduction in toxicity, superior ability to metabolize starch and functional differences in flavor-associated genes, suggesting that, while plant and animal domestication was driven primarily by genetic alterations in developmental pathways, extensive changes of metabolism dominated microbe domestication. The third and final facet focuses on comparative genomic analyses of tandem repeats in coding and non-coding regions. Examination of coding regions revealed that the presence and copy number of tandem repeats was highly variable within and between species and overrepresented in genes with specific functions key to the fungal lifestyle. In non-coding regions, my study found little evidence supporting the longstanding, yet sparsely tested, hypothesis that tandem repeats are a universal source of rapid phenotypic variation.

Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize olfactory and temperature cues in host-seeking behaviors that underlie their vectorial capacity. By using a combination of molecular and physiological approaches, we have studied the three head appendages of female An. gambiae mosquitoes, the antenna, the maxillary palp and the proboscis, and have characterized their respective functions in olfaction or thermoreception that might impact the host-seeking behavior of An. gambiae. Specifically, we have described three types of sensilla, the labellar T2 sensilla, the palpal capitate pegs and the antennal small coeloconica sensilla, and have characterized their responses to olfactory or temperature stimulation and have identified the underlying molecular and cellular components of these sensilla. These results facilitate the understanding of how An. gambiae mosquitoes sense human hosts that might be exploited to compromise their ability to transmit malaria.

Arguably, the process of adaptive ecological speciation and radiation has been the most important mechanism in contributing to the worlds immense biological diversity. Initial theoretical treatments of adaptive speciation made special mention of species interactions, namely interspecific competition, as an important source of divergent natural selection that ultimately contributed to the formation of novel ecological and species diversity. However, accumulating evidence suggests that mutualistic associations, especially those between microbes and insects, may be important in the process of adaptive speciation, as mutualists can act as sources of important phenotypic variation. In this dissertation, I investigate aspects of the interaction between the goldenrod-galling midge, <i>Asteromyia carbonifera</i>, and its fungal symbiont, <i>Botryosphaeria dothidea</i>, and attempt to relate the findings to ongoing evolutionary diversification within the species. First, a verbal theoretical treatment of how microbial mutualists could affect the process of adaptive speciation is put forth, using insects and their diverse microbial associates as examples. Second, I show by sterol-profile analysis that <i>A. carbonifera</i> appears to feed exclusively on its fungal associate, which may be important in opening nutritional ecological opportunity. Finally, I show that while the fungal associate mediates the observed phenotypic variation in gall morphology, it is the midge itself that is directly responsible for the ecologically important phenotypic variation. Moreover, the fungal associate does not exhibit the typical evolutionary signatures of a heritable mutualism. These data suggest that, for <i>Asteromyia carbonifera</i>, the fungal symbiont may indeed be a crucial player in the ongoing evolutionary radiation of its host, but the association does not appear to be a typical host-symbiont coevolutionary interaction.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant human disease caused by the expansion of a tetranucleotide repeat in the first intron of the ZNF9 gene. While multiple studies link nucleotide expansions such as those found in the ZNF9 gene to a variety of other human diseases, it is unclear whether the expanded repeat results in a misregulation of ZNF9. This work focuses on the molecular function of the ZNF9 protein and what role aberrant ZNF9 expression plays in DM2 pathology. ZNF9 activates cap-independent translation of ornithine decarboxylase (ODC) through a direct interaction with the 5 untranslated region of the ODC mRNA. This activity is reduced in myoblasts from DM2 patients, suggesting that nucleotide expansions at the DM2 locus result in a misregulation of the ZNF9 protein. An ortholog of ZNF9 in Saccharomyces cerevisiae, Gis2p, shows striking conservation in biochemical interactions and can activate translation of the ODC mRNA in mammalian systems, providing an alternative system for studying ZNF9 activity and function. Finally, mass spectrometry-based proteomic analysis of DM2 mouse models reveals that a large number of skeletal muscle regulatory proteins show aberrant expression as a result of a loss of ZNF9 activity. Together, this work provides evidence that ZNF9 acts as a regulator of the essential cellular process of translation and that DM2 may result due in part to defects in the expression or regulation of this protein.

Fragile X syndrome, resulting from the loss of function of the hFMR1 gene, is the most common heritable cause of intellectual disability. The human genome also encodes two closely related paralogs: hFXR1 and hFXR2. Drosophila that lack the dFMR1 gene recapitulate FXS-associated molecular, cellular and behavioral phenotypes. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. All three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other non-neuronally. It has been long hypothesized that the phosphorylation of serine 500 in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wildtype (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1). At the molecular and cellular levels, the phosphomimetic is able to rescue elevated protein levels and architecture overgrowth phenotypes, whereas the dephosphomimetic phenocopies the null condition. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.

The existence of anatomical differences along the left-right (L-R) axis in the brain and visceral organs is an evolutionarily conserved feature of the vertebrate lineage. Although much is known about the molecular pathways that lead to the development of visceral organ asymmetry, we are just starting to understand the molecular processes that lead to the development of brain laterality. The zebrafish epithalamus, which includes the pineal organ and a left sided accessory organ called the parapineal organ, offers a powerful model to study the events leading to the generation of brain asymmetry. A chemical mutagenesis screen was performed to identify mutants that disrupt laterality in the epithalamus. The from beyond (fby)/tbx2b mutation causes a brain-specific defect in laterality including a decrease in the total number of specified parapineal cells and an inability of parapineal cells to migrate to the left of the pineal organ. Time-lapse imaging of wild-type parapineal cells show several cells emerging from the pineal complex anlage and migrating as a group in a leftward direction. Similar time-lapse imaging of fby mutants shows that this leftward migration does not occur. Further, ablation of some parapineal cells in wild-type embryos show that reduced cell number does not prevent migration of the remaining cells in fby mutants. Characterization of a hypomorphic allele of tbx2b, lots-of-rods (lor), further supports the hypothesis that specification and migration defects found in tbx2b mutants are separable events that require different doses of tbx2b. Currently, the transcriptional regulators of tbx2b in the pineal complex are unknown; however a previous report had suggested that Floating Head (flh) protein controls tbx2b transcription in the epithalamus. Our studies suggest that this is not the case, and that Flh and Tbx2b act in parallel pathways. Fate mapping, in conjunction with efforts to identify upstream regulators and downstream targets of tbx2b activity in the developing pineal complex, will help determine the mechanism of parapineal organ formation.

DNA polymerase ¦Á-primase (pol-prim) plays a central role in eukaryotic DNA replication, initiating synthesis on both the leading and lagging strand DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68, that is thought to regulate the complex. In the Simian Virus 40 (SV40) replication model, the p68 subunit is required for primosome activity and interacts directly with the viral helicase T antigen (Tag), suggesting a functional link between Tag-p68 interaction and primosome activity. To explore this link, I first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the Tag helicase domain. The solution structure of p68N was determined by NMR spectroscopy in the Chazin lab. The putative Tag-interacting surface on p68N was mapped to a hydrophobic patch surrounded by negative charges. Structure-guided mutagenesis of p68 residues in the interface diminished Tag-p68 interaction and primosome activity of the SV40 replisome. The p68N-docking site on Tag was identified using structure-guided mutagenesis of the Tag helicase surface. A charge reversal substitution in Tag specifically disrupted p68N binding, and hence the SV40 primosome activity. These results collectively suggested that the Tag-p68 interaction is vital for SV40 primosome function. A model is presented for how this interaction regulates primosome activity of the pol-prim complex, providing significant insights into the mechanism of eukaryotic DNA replication initiation.

SPECIATION AND DIVERSIFICATION IN THE NORTH AMERICAN TIGER BEETLES OF THE CICINDELA SYLVATICA GROUP: MORPHOLOGICAL VARIATION AND AN ECOPHYLOGEOGRAPHIC APPROACH DANIEL PAUL DURAN Dissertation under the direction of Professors Daniel J. Funk (2004-2009) and Charles K. Singleton (2009-2010) In this dissertation, I addressed three fundamental questions in evolutionary ecology: 1) What factors promote population differentiation and speciation? 2) What are the determinants of species ranges? and 3) What are the principal causes of phenotypic variation? I used multiple phylogenetic, population genetic, and coalescent-based molecular analyses in conjunction with GIS-based environmental niche modeling and statistical analyses of phenotypic characters to test hypotheses concerning the evolutionary history and ecology of a group of North American tiger beetles. Using a congeneric phylogeography approach, I sampled intensively from all the nominal species and subspecies of the North American Cicindela sylvatica group. None of the nominal species were found to be monophyletic with respect to mtDNA. Analyses supported multiple underlying causes for the species-level polyphyly, including cases of polymorphism, hybridization, and incomplete lineage sorting. Abiotic factors were shown to be primarily limiting species distributions. These same environmental factors were also significantly predictive of color pattern variation in C. longilabris. This integrative multidisciplinary approach can serve as a model for future studies. Each series of investigations reciprocally informed the others, leading to a more robust set of conclusions about the causes of differentiation and speciation in these taxa. The results of this dissertation also have important consequences for systematic research in general, especially for the disciplines of molecular systematics or molecular taxonomy (Blaxter and Floyd 2003; Tautz et al. 2003; Blaxter 2004), and DNA barcoding (Hebert et al. 2003 a, b). In addition to these larger issues, there are more specific systematic and taxonomic implications for understanding the causes for the patterns of distribution of the North American Cicindela sylvatica group and consequences for future tiger beetle research.

Circadian rhythms are intrinsic biological rhythms that have a period close to 24 hours. Prokaryotic cyanobacteria are the simplest organisms that show robust circadian rhythms, and are the only organisms that have a circadian rhythm reconstituted in vitro in test tubes in the presence of ATP. This thesis is focused on studying the mechanism of the core circadian clockwork in the cyanobacterium Synechococcus elongatus PCC 7942. In this dissertation, I explore the mechanism of the circadian rhythm in cyanobacteria with both in vitro and in vivo studies. I elucidate the essential pattern of dynamic protein- protein interactions among three identified core clock Kai proteins, and define the relationship between the traditional TTFL (transcription translation feedback loop) model and the PTO (post translational oscillator) model within this strain. In addition, a KaiC monomer exchange phenomenon is studied to further investigate the robustness of the rhythm.