Maternally transmitted microbes that infect host germ cells are perfectly poised to influence animal genome evolution, either directly through horizontal gene transfer or indirectly through selection for host genetic variants that control symbiont proliferation. In this dissertation, both of these scenarios are examined using the obligate, intracellular bacteria Wolbachia as a model for maternally-transmitted bacteria because of its widespread distribution across 40-50% of all arthropod species and its localization to the maternal germ line cells. In the first project, whole-genome sequencing of uninfected Chorthippus parallelus grasshoppers revealed that large regions of DNA from two different supergroups of Wolbachia had horizontally transferred into the grasshopper genome. While horizontal gene transfer of Wolbachia DNA to a eukaryotic host is common given Wolbachia?s proximity to the germ line genome, this is the first known example of two divergent Wolbachia strains contributing DNA to the same host genome. Furthermore, Wolbachia inserts were present in almost all of the grasshopper chromosomes and often differed between closely-related C. parallelus subspecies, indicating that horizontal gene transfer from Wolbachia is an unusually dynamic process in grasshoppers. In the second project, a forward genetic screen was conducted to find host genomic regions responsible for regulating an 80-fold difference in Wolbachia titers between two closely-related species of Nasonia parasitoid wasps (N. vitripennis and N. giraulti). Quantitative trait loci analyses and hybrid introgressions identified two genomic regions, one each on chromosomes 2 and 3, that act additively through a maternal effect to suppress Wolbachia titers in N. vitripennis. Thirty-three significantly differentially expressed genes are present in these regions, several of which function in pathways important for host control of intracellular bacteria including immunity, autophagy, and cell-to-cell trafficking. Additionally, staining of Nasonia ovaries with a nucleic acid dye revealed that N. vitripennis may keep Wolbachia out of developing oocytes by sequestering them in the neighboring nurse cells. Candidate genes that are overexpressed in N. vitripennis, such as trichohyalin, or those involved in Wolbachia trafficking, such as kinesin, are currently being evaluated using RNAi for their role in host regulation of Wolbachia titers and transmission.

Glycosylation is the most common post-translational modification to proteins, involving the addition of chained sugars to regulate folding, localization and intermolecular interactions. Glycosylated proteins are most heavily concentrated on the extracellular side of cellular membranes, and most secreted proteins are glycosylated. Both of these glycoprotein classes are critical for cell-cell interactions, particularly during metazoan development. The nervous system is enriched for glycoproteins, and neurons appear dependent on glycosylation in the regulation of synapse structure and function. This thesis tests glycosylation roles at the synapse by analyses of two genes at the Drosophila neuromuscular junction (NMJ); mgat1 required to produce hybrid and complex glycan branches, and pmm2 required to produce all N-linked glycosylation. Loss of either gene results in defective synaptic glycosylation, and similarly overelaborated NMJ architecture and elevated neurotransmission. Moreover, both mutant conditions cause aberrant trans-synaptic signaling that normally directs the recruitment of synaptic proteins required for synaptogenesis and neurotransmission. Thus, synaptic glycosylation strongly modulates the trans-synaptic signaling that in turn drives the recruitment synaptic proteins that mediate of structural and functional synaptogenesis. This thesis produces a new genetic model for the heritable Congenital Disorders of Glycosylation (CDG) disease state CDG1a (a.k.a. PMM2-CDG), producing an avenue for the development of treatments and therapeutic interventions.

Non-muscle myosin II (MyoII) contractility is important to the regulation of many cellular processes, including migration. The small GTPase Rho has been shown to regulate MyoII contractility, but the role of other GTPases, such as Rac, in modulating contractility is not well understood. In this study, we show that activation of Rac by the guanine nucleotide exchange factor (GEF) Asef2 increases MyoII contractility to impair cell migration on two-dimensional (2D) type I collagen. Knockdown of endogenous Rac using short hairpin RNAs (shRNAs) or treatment of cells with the Rac-specific inhibitor NSC23766 results in a significant decrease in the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Furthermore, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. Because three-dimensional (3D) matrices more closely mimic the physiologic environment of cells, we investigated the role of Asef2 in regulating migration and MyoII activity in 3D environments. For these studies, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that Asef2 inhibits cell migration in 3D type I collagen matrices, and treatment of cells with blebbistatin abolishes the Asef2-mediated decrease in migration. Moreover, Asef2 enhances MyoII activity as shown by increased S19 phosphorylation, and treatment with NSC23766 abolishes the Asef2-promoted increase in active MyoII. Collectively, these results indicate that Rac activation, promoted by Asef2, is critical for modulating MyoII activity and cell migration in both 2D and 3D environments.

Carbonic anhydrases (CAs) are zinc-metalloenzymes that interconvert two inorganic carbon (Ci) species, CO2 and HCO3-. In Arabidopsis thaliana, there are eight Alpha-CA genes, six Beta-CA genes, three Gamma-CA genes, and two Gamma-CA-like genes. The majority of CA research in plants has focused on finding a link between CA activity and photosynthesis rates. Since the CA genes are expressed in different plant tissues and multiple CA isoforms are distributed among various organelles of the plant cell, I hypothesize that CAs facilitate CO2 diffusion among cell compartments and maintain Ci pools for carbon-requiring reactions by interconverting CO2 and HCO3-. This thesis focuses on the Alpha-CAs and Beta-CAs of Arabidopsis and how they may affect various reactions throughout the plant. CA T-DNA insertion lines were used to determine if removing one or more CAs from Arabidopsis affects the plant growth. The Beta-ca5 single mutant and Beta-ca2Beta-ca4, Alpha-ca1Beta-ca4, and Alpha-ca2Beta-ca4 double mutants show different growth phenotypes. The Beta-ca2Beta-ca4 plants were smaller in size and chlorotic in their younger leaves under low CO2 conditions, but showed improved growth in high CO2 conditions. The growth of the Beta-ca5 single mutant was severely stunted in ambient CO2 conditions and high CO2 partially rescued wildtype growth in the Beta-ca5 plants. The Alpha-ca1Beta-ca4 and Alpha-ca2Beta-ca4 double mutants were slightly smaller than wildtype plants in low CO2 conditions. Interestingly, it seems the reduced growth of the Beta-ca5 single mutant and Beta-ca2Beta-ca4 double mutant plants was not linked to deficiencies in photosynthesis rates but rather may be required for other carbon requiring reactions. These results suggest that CAs are playing more complex roles in plants than once thought and that the various isoforms are affecting different carbon-requiring pathways.

The causes and implications of differences in geographic variation across species are generally poorly understood, but comparative studies have the potential to provide better understanding of what factors predispose species to undergo population divergence and whether population divergence has lasting evolutionary impacts. Here, I examined geographic variation in birds using molecular data from across the genome. I characterized genetic diversity, estimated population history, and tested for impacts of landscape history as well as ecological traits on genetic parameters. I found evidence that diverse historical processes have led to present-day genetic variation in Neotropical bird species, including divergence, population expansion, migration, and gene flow. Genetic diversity and historical processes differed across species, and some of these differences were associated with habitat. Birds of upland forest had greater genetic diversity, higher divergence between populations, and deeper population histories than birds of floodplain forest in the Amazon. This may result from higher dispersal in floodplain species, recent population expansion in or colonization of floodplain habitats, or persistent demographic differences between habitats. I also found that rates of population divergence within species predicted rates of speciation in their ancestral lineages. This result suggests that traits that predict population divergence within species, such as their habitat associations, will impact their diversification over long evolutionary timescales.

The relationships between the European species of Vulpia were investigated by interspecific hybridisation and consequent meiotic analysis, hybrid fertility and morphological survey of the F1 hybrids. The relationship between the genus Vulpia and the Festuca rubra aggregate was investigated by the examination of both naturally occurring and artificially produced intergeneric hybrids. Embryo culture was used for both interspecific and intergeneric crosses and the germination of the hybrid embryos by this method proved successful. Of 47 cross combinations (including reciprocals) attempted hybrid plants were obtained for 20 different crosses. Crossability and hybrid fertility were not found to be obvious indicators of taxonomic distance and unilateral incompatibility was found both in crosses with a self-incompatible species as the female parent and a self-compatible species as the male parent and in crosses where both parental species were self-compatible. Analysis of bivalent pairing at meiosis revealed genome relationships in hybrids between diploid species. However no conclusions could be made as to the possible ancestors of tetraploid V. fasciculata. Artificial hybrids were made between V. sicula, the presumed link species between the annual species of Vulpia and the perennial fescues, and F. rubra and the meiotic analysis of the F1 hybrid revealed similarities between at least part of the V. sicula genome and a genome of F. rubra. Conclusions as to genome homology could not be made from the cytological analysis of the other intergeneric hybrids.

Regulatory elements are DNA sequences which have specialized activities that coordinate the functions of the genome. Promoters, enhancers, locus control regions, boundary elements (or insulator elements) are examples of DNA sequences that have regulatory properties. In transgenic assays insulator elements have been shown to block communication between regulatory regions, such as enhancers and promoters, when placed between these sequences and also protect genes from position effects when bracketing them, thereby affecting gene expression. Insulator sequences are bound by insulator proteins that direct the function of these sequences. One such insulator protein is the Boundary Element Associated Factor-32 (BEAF-32), a 32 kDa protein which was originally found to bind to the scs insulator sequence in the 87A heat shock locus of the Drosophila genome. BEAF-32 has two isoforms: 32A and 32B. BEAF was immunolocalized to numerous binding sites across the Drosophila genome. This was substantiated by various genome-wide mapping experiments, which have identified from 1800 to 6000 BEAF binding sites across the genome. Hence, BEAF-32 likely plays an important role in chromatin organization and gene regulation in combination with other proteins in the nucleus. However, it is not clear how BEAF-32 affects genome organization and gene regulation. We characterized essential domains in the BEAF-32 protein and identified protein partners, some of which include Transcription Factors (TFs). We further mapped the interaction regions inside BEAF and these TFs. We then attempted Fluorescent Recovery After Photobleaching (FRAP) to assess the dynamics of BEAF-32 on polytene chromosomes and also observed banding patterns, with the help of fluorescent protein labels, and evaluated its behavior during mitosis in early embryos. Finally, results obtained with BEAF prompted us to test for physical interactions between various insulator proteins and to check contradictory reported results from the literature to document interactions.

DNA double-strand breaks (DSBs) are the most lethal form of DNA damage; a single DSB, if left unrepaired, can cause cell death. Inaccurately repaired DSBs are sources of mutations, chromosomal rearrangements and genome instability. Telomeres, TG-rich nucleoprotein structures at the termini of eukaryotic chromosomes, protect chromosome ends from nucleolytic degradation and distinguish chromosome ends from those generated by DSBs. In most eukaryotes, proper telomere maintenance requires a specialized reverse transcriptase, telomerase, that utilizes an enzyme-associated RNA as template for new telomere synthesis. Increasingly, telomere-associated proteins are being recognized as playing roles in the repair of internal DSBs. At endonuclease-induced DSBs immediately preceded by ectopic, short telomere seeds, the telomeric single-stranded DNA binding protein Cdc13 recruits telomerase, promoting the addition of new (de novo) telomere sequences to the telomere seed. However, spontaneous DSBs occur very infrequently and as such, are rarely expected to occur immediately adjacent to telomere-like seed sequences. An experimental system in which a DSB is induced at a distance (~3 kb) from endogenous Sites of de novo Telomere Addition (SiRTAs) in budding yeast was utilized as a more representative model of de novo telomere addition following spontaneous DSBs. These telomere-like SiRTAs incurred high frequencies of de novo telomere addition relative to flanking sequences. Additionally, de novo telomere addition at these SiRTAs required a bipartite structure in which Cdc13 binding to a stimulatory (Stim) sequence strongly stimulates telomere addition at a nearby target (Core) sequence. The role of yet another budding yeast telomere-associated protein, Rif1, in DSB repair was also explored using an experimental system of an endonuclease-induced DSB that is primarily repaired by imprecise non-homologous end joining (NHEJ). Wild-type repair junctions contained only deletions. However, rif1â repair junctions contained relatively smaller deletions, as well as insertions, indicative of ends undergoing less resection in the absence of Rif1. A rif1 mutant lacking the protein phosphatase-1-interacting domain phenocopies the rif1â mutant, implicating this domain in Rif1âs role in regulating the fidelity of DSB repair.

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No description available.