Sex is a major challenge to evolutionary theory, because of the apparent paradox of its ubiquity among multicellular eukaryotes with the substantial evolutionary costs associated with it. The past four decades have seen the development of a large and robust collection of hypotheses purporting to explain the benefits of sex and reconcile this paradox, while empirical research testing the predictions of these hypotheses in nature has only begun to gather momentum more recently. Neochlamisus leaf beetles are one such natural system with great potential for study of the evolutionary tradeoffs of sexuality and asexuality thanks to the prevalence of gynogenetic asexuality within the genus. Gynogenesis is a form of asexual reproduction in which females require insemination to stimulate the development of unreduced eggs into clonal offspring with no paternal genetic contribution. Gynogenesis requires the coexistence of asexual females with females and donor males of the same nominal species, allowing highly controlled comparisons of ecologically and biologically similar sexual and asexual animals. In this thesis, I present a new mitochondrial DNA dataset consisting of diverged sexual and asexual lineages of Neochlamisus and describe the phylogenetics and patterns of molecular evolution observed in each lineage, with explicit regard to reproductive mode. I then test two a priori predictions of differences in molecular evolution between the sexual and asexual lineages related to reproductive mode. My results are consistent with a higher rate of accumulation of putatively harmful nonsynonymous mutations in the asexual lineage, and with a selective sweep of the mitochondrial genome in the sexual lineage driven by cytoplasmic incompatibility-inducing strains of the intracellular bacterial parasite Wolbachia.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol 3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant negative or deleterious gain of function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain synaptic functional efficacy.

The adult vertebrate forebrain is responsible for a diverse set of behaviors, and as such exhibits complex anatomy. This complexity is generated during embryogenesis whereby a specific spatiotemporal sequence of transcriptional and signaling programs promotes specification of different cell types based on the location and developmental potential of progenitors. Six3 is one such transcription factor that exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly, a forebrain malformation resulting, at least in part, from abnormal telencephalon development. However, Six3s roles in telencephalon patterning and differentiation are not well understood. The zebrafish genome contains three Six3-related genes facilitating analysis of different partial loss-of-function combinations. I analyzed zebrafish embryos deficient in two of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. Telencephalon forms in six3b;six7-deficient embryos, however ventral telencephalic domains are reduced and dorsal domains are expanded. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog signaling in telencephalon development. Unlike in mice, Hedgehog signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hedgehog signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hedgehog-independent role for Six3 in telencephalon patterning. Additional investigations into the interactions between Six3 and known telencepnalon patterning genes showed that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of Wnt/β-catenin pathway. As Six3-related genes are expressed broadly in prechordal mesoderm and anterior neuroectoderm, transgenic zebrafish were generated to identify the spatial requirement for Six3 function in telencephalon patterning. My studies help define the cellular mechanisms of Six3-mediated dorsoventral patterning in telencephalon and present a novel genetic mechanism by which Six3 regulates this process.

Recent empirical studies conducted in disparate ecosystems have shown that greater species diversity has positive effects on ecosystem functioning; however, other studies have found neutral or sometimes negative results. It is still unclear why the relationship between biodiversity and functioning varies among studies, but perhaps, investigating this relationship across spatial and temporal scales will lead to further understanding. One theory predicts that local niche complementarity among species (the partitioning of species based upon niche differentiation) is predicted to positively affect local ecosystem functioning at the local spatial scale. However, more recent theory predicts that greater local diversity may hinder local ecosystem functioning when diversity is enhanced through regional processes. I suggest community assembly as a way to incorporate both the local and regional processes that determine biodiversity and its consequent effects on ecosystem functioning. From this, I propose a hump-shaped relationship between diversity and ecosystem functioning at local spatial scales, but a linear increase of functioning with diversity at regional spatial scales. Thus, species diversity may have different effects on ecosystem functioning across different spatial scales. Species diversity may affect ecosystem functioning differently across time as environmental conditions shift. Through integrating recent theoretical models in ecosystem ecology and empirical examples of food-webs in community ecology, the effects of herbivore diversity on ecosystem functioning (grazing of primary producers) were examined under unchanged (no nutrients added) and changed (nutrients added) environmental conditions. I found that communities with higher species richness and diversity did not significantly differ from lower diversity communities in grazing intensity in the unchanged environments. However, higher diversity communities did have a significant effect on the biomass of primary producers in the nutrient enriched environments, while lower diversity communities did not. This empirical study showed that the functioning of local communities is dependent on the environmental conditions present in the habitat. Overall, this investigation found that the relationship between species diversity and ecosystem functioning may be dependent on spatial scale and environmental changes over time.

The effects of nutrient input into aquatic systems has been studied frequently; typically, these studies report an increase in algal biomass and a decrease in species diversity in response to an increase of nutrients. However, it is not clear why similar aquatic communities will respond differently to nutrient additions of similar magnitudes, resulting in alternative communities. Because variance in natural ecosystems is pervasive, perhaps it is this variability that helps determine the final community. I proposed that the total amount of nutrient input and the variability of nutrient input would affect the abundances and composition of species. A natural survey was conducted to measure the variable levels of nutrients in several aquatic systems. Experimental ponds were used to test the effects of variable rates and timing of nutrient inputs upon an aquatic community; experimental treatments manipulated the total amount of nutrient input (high v. low), the rate of nutrient input (annually, monthly or weekly), the timing of the nutrient input (early v. mid- season), and the trophic status at which these treatments were imposed (mesotrophic v. eutrophic). The effects of the variability of nutrient input was at least as important as the total amount of the nutrient input. There were large impacts upon species diversity, abundances and composition. Although these effects were manifested in many trophic groups, the response to the variability was most strikingly found within the primary producers, which showed large shifts in abundance and composition.

Mitotic segregation defects such as multipolar spindles, anaphase bridges, and micronuclei have long been observed in cancer cells, but it is not known whether these defects lead to aneuploidy or even contribute to tumorigenesis. We visualize living oral squamous carcinoma cells with stable expression of GFP-histone H2B fusion. Expression of this fusion protein labels chromosomes clearly and does not disrupt the cell cycle, alter the doubling time or produce any defects previously unseen in fixed cells. These carcinoma cells survive the formation of anaphase bridges and micronuclei and complete a second cell division in the same amount of time as unaffected cells. Micronuclei were formed after every division that contained an anaphase bridge in cells we examined. Most often, each daughter cell contained a micronucleus. These results suggest that the chromosome breaks at multiple points along its length and breaking may not be due to a "tug of war" between spindle poles. The movement of micronuclei was very dynamic compared to the nuclei during interphase and micronuclei do not appear to be transcriptionally active. Using long-term live cell imaging we were also able to observe the fate of these cells through two divisions and have determined the length of each phase of mitosis. Anaphase bridges and lagging metaphase chromosomes both lengthen mitosis, suggesting that the mitotic spindle checkpoints are at least partially active in cells. The mitotic delays occurred during metaphase in these defective cells. We have also analyzed centrosomal components including the mitotic apparatus protein, NUMA. No correlations were found between protein expression of NuMA and gene amplification or segregation defects. In summary, we have shown that cells continue to proliferate after the occurrence of mitotic defects and these defects contribute to chromosomal instability.

Control of directionality in integrase-mediated site-specific recombination reactions is achieved by an architectural change fashioned by a class of accessory proteins know as recombination directionality factors (RDFs). In the mycobacteriophage L5 system, no RDF had been previously identified. In the course of this work, the gene, 36, was identified using in vivo screens and shown to play this role. The protein was over expressed using an E. coli expression system and then used to create an in vitro excision reaction assay. Initial work was done in characterizing the excision reaction including supercoiling and host factor requirements. Further analysis has shown that the protein binds specifically to a region within the left side of attP and attR. Once bound, complexes are formed that inhibit integration in the case of attP and stimulate excision when attR is present. Additional work was done to increase the utility of the L5 based integrating vector system, which has been commonly used to create stably integrated single copy transformants. The system lacked an effective means to recover DNA for high copy replication or curing of strains containing inserted DNA. With the identification of the L5 xis gene, we have been able to develop systems that allow the recovery of DNA into E. coli and curing strains of the integrated DNA. The third part of this work involved the characterization of the RDF class of proteins. Since this class of proteins is composed of a diverse group of small proteins, no previous attempt had been made to characterize them. Extensive data mining yielded a collection of 63 putative or known RDFs. Further analysis of sequence data, chemical characteristics and other known properties argues that this class of proteins has evolved from multiple ancestral origins.

Lysogeny is a defining feature of temperate bacteriophages. Temperate bacteriophages are able to establish lysogeny by integrating into the host chromosome or extrachromosomally, as a plasmid-like prophage in the host cytoplasm. In either case, host factors are involved in both the establishment and maintenance of lysogeny. Mycobacteriophage L5 forms an integrated prophage through the action of a phage-encoded tyrosine recombinase. L5 integrase (Int) binds to the phage attachment site, attP, and the bacterial attachment site, attB. Int binds attP bivalently by binding to the core, where strand exchange occurs, and to arm-type binding sites that flank the core. A host-encoded DNA binding protein, mIHF, is required for recombination and binds between the core and arm-type binding sites of attP. mIHF is thought to catalyze the bending of attP required for bivalent binding between the core and arm-type binding sites. attP core consists of a seven base pair overlap region flanked on either side by imperfect inverted repeats that make up the recombinase binding elements or RBEs. The RBEs were shown to be essential for core binding by creating attPs with mutations in the RBEs. When both of the RBEs are mutated Int can neither perform recombination, nor create specific complexes that involve core binding. When the right side RBE is wild type with mutant left side RBE, recombination can occur, and complexes involving core binding are observed. However, Int is unable to catalyze recombination when the left side RBE is wild type and the right is mutated, indicating that contact must be made with attP on the right side of core for a stable Int/attP core complex to be formed. The middle domain connects the two outer domains, but its function in recombination is not well understood. To determine the function of the middle domain, mutations were made at conserved residues in the middle domain of Int, and these mutants were characterized. These mutants are able to catalyze recombination, but do not form the recombinagenic complexes observed by wild type Int. Increasing the mIHF concentration in these reactions augments the recombination efficiency and stabilizes recombinagenic complexes that involve Int/core binding.

DNA methylation in mammals is involved in several essential processes including X chromosome inactivation, genomic imprinting, and host defense against mobile genetic elements. How methylation is targeted to specific sequences in the germ line and how specific methylation patterns are maintained during development is not fully understood. Genomic methylation is established in the gamete, decreases dramatically during preimplantation development, and is re-established after implantation of the blastocyst. However, the methylation present at imprinted loci is specifically maintained during preimplantation development. Imprinted genes are located in clusters, and within each gene cluster parent-of-origin specific expression is governed by an imprinting center (IC). The ICs of the maternally imprinted murine Snrpn, Kcnq1, and Igf2r gene clusters coincide with their differentially methylated domains (DMDs). We have shown that specific DMD sequences are required to establish differential methylation at an imprinted locus. Hybrid transgenes were generated using a non-imprinted derivative of the imprinted RSVIgmyc mouse transgene, Ig/myc, and sequences from endogenous imprinted gene DMDs. Addition of specific DMD sequences to the Ig/myc transgene restored its imprinting. Only the tandem repeats found within the Snrpn, Kcnq1, and Igf2r DMDs were capable of establishing maternal-specific transgene methylation. These experiments have also shown that the methylation on imprinted gene DMD sequences is specifically maintained during the early stages of preimplantation development. These results clearly demonstrate the importance of tandem repeats in the process of genomic imprinting. DNA methylation is also critical for silencing intracisternal A particle (IAP) transposition within the genome. It is thought that maintenance of IAP element methylation during preimplantation is critical to repress IAP element transcription and transposition. The methylation of IAP element long terminal repeat (LTR) sequences was analyzed at the blastocyst stage of preimplantation development using the bisulfite genomic sequencing technique. These experiments have shown that methylation is maintained on the majority of IAP elements at the blastocyst stage of preimplantation development. However, the methylation on specific IAP elements is completely lost at this time, including the methylation of single IAP element LTRs.

The site-specific recombination systems of bacteriophages and other mobile elements fall into two categories that are named for the integrase protein that catalyzes integration and excision of the phage genome. These integrases utilize either a tyrosine or a serine residue to carry out the nucleophilic attack of the DNA. In many cases the directionality of the reaction, that is whether the enzyme catalyzes integration or excision, is determined by an additional phage encoded protein referred to as RDF, or recombination directionality factor. Two prophage-like elements, phiRv1 and phiRv2, were found through sequencing Mycobacterium tuberculosis strain H37Rv. These are absent from the vaccine bacillus M. bovis BCG, and are often found in virulent strains of M. bovis and M. tuberculosis. Through the work presented here, one of these elements, phiRv1, was found to encode an active recombination system, with a serine integrase and RDF. The phiRv1 element is found within a degenerate repeated element, REP13E12, which is present in seven non-identical copies in M. tuberculosis and M. bovis BCG. In vivo studies have revealed that four of the seven 13E12 elements can serve as integration sites (attBs) for a plasmid carrying a reconstructed attP and integrase, and that multiple integrations can occur. The fast growing saprophyte, M. smegmatis, also supports integration, although inefficiently. In M. smegmatis, the phiRv1 plasmid integrates into at least two 13E12 repeats that are quite different from those found in M. bovis BCG and M. tuberculosis. Inefficiency is overcome by providing M. smegmatis with an attB site from BCG. These integrated plasmids are stable, and excision occurs in the presence of the phiRv1 RDF encoded by Rv1584c. In vitro assays were developed for both integration and excision. All the substrate site requirements are relatively small. Integration occurs slowly but efficiently in the presence of excess attB or on an intramolecular attP-attB plasmid. In the presence of RDF integration is inhibited and excision is stimulated. The RDF binds to a specific sequence in both attB and attL, although the way in which it functions may be through protein-protein interactions with integrase.