AEBP2 is a zinc finger protein that has been shown to interact with the mammalian Polycomb Repression Complex 2 (PRC2). I characterized this unknown protein and tested its potential targeting roles for the PRC2. AEBP2 is an evolutionarily well-conserved gene that is found in animals ranging from flying insects to mammals. The transcription of mammalian AEBP2 is driven by two alternative promoters and produces multiple transcripts that give rise to at least two isoforms of the protein. These isoforms show developmental stage-specific expression patterns: the larger adult-specific form (52 kDa) and the smaller embryo-specific form (31 kDa). The AEBP2 protein binds to a DNA-binding motif with an unusual bipartite structure, CTT(N)15-23cagGCC with lower-case base pairs being less critical. A large fraction of AEBP2's target loci also map closely to the known target loci of the PRC2. In fact, many of these loci are co-occupied by the two proteins, AEBP2 and SUZ12. This suggests that AEBP2 is most likely a targeting protein for the mammalian PRC2 complex. To investigate the in vivo roles of this protein, a mutant mouse line with disrupted Aebp2 transcription has been generated. Breeding experiments demonstrated embryonic lethality in the Aebp2-mutant homozygotes, but survival of the heterozygotes to adulthood with fertility. In developing mouse embryos, Aebp2 is expressed mainly within cells of neural crest origin, such as the dorsal root ganglia, and facial cartilages and bones. In addition, many heterozygotes display a set of phenotypes, including enlarged colon and hypopigmentation, similar to those observed in human patients with Hirschsprungs disease and Waardenburg syndrome. These phenotypes are caused by the absence of the neural crest-derived ganglia in hindguts and melanocytes. Additional analyses further confirmed changes in the expression and methylation levels of H3K27me3 on the genes involved in the development of the neural crest cells in the Aebp2 heterozygotes. Overall, these results suggest that Aebp2 may regulate the development of the neural crest cells through the PRC2-mediated epigenetic mechanism.

The sale of eastern oysters (Crassostrea virginica) is a multi-million dollar industry in the United States. Triploid production of oysters helps to alleviate the decline in sales during summer months that normally results from the loss of flavor and texture when diploid oysters are spawning. Research to improve the ease of triploid production is critical for the expansion of oyster hatcheries producing triploids. The goals of this research were to determine the period of time when triploid oysters are most fertile and to determine if prostaglandin E2 (PGE) plays a role in oocyte maturation of C. virginica. The results of this research will assist in creating triploid broodstock for tetraploid production. Triploid oysters were found to reach full gonadal maturity as well as spawn, however at lower percentages than diploid oysters. In this study, diploid oysters peaked in maturity from April 18-May 07. Triploid oysters peaked in maturity in early February and had two smaller peaks in early June and mid August. This suggests that triploid development is shifted from diploid development and should be spawned either earlier or later in the year than diploids. Oysters with oocytes in early development (stage II) continually increased PGE titers through stages III and IV. Once oysters were ready for spawning in stage V, titers decreased in value. This correlation of PGE titers with gonadal stage suggests that PGE plays a role in the early maturation phase of oocytes. PGE titers were not significantly different when compared to 2N and 3N cohorts or amongst gonadal stages. The lack of significance may be due to high variability as a result of random sampling.

Resprouting and reseeding shrubs are predicted to be restricted to habitats with long and short return intervals of natural disturbances, respectively. Nonetheless, resprouters and reseeders co-occur along Gulf of Mexico coastal ecoclines where lightning-ignited fires were historically frequent. In this collection of studies, I examine fire responses, habitat associations, distribution limits, and seedling recruitment of <i>Hypericum</i> species along ecoclines in pine savannas of the St. Joe Bay State Buffer Preserve in Gulf County of Florida. In this area, ecoclines are characterized by topographic, soil moisture, and fire heterogeneity gradients. <i>Hypericum microsepalum</i>, an obligate resprouter, primarily resprouts following disturbances that remove aboveground stems. It is associated with upland, dry habitats that frequently burn, but can survive over a wide range of habitats along ecoclines. <i>Hypericum chapmanii</i>, an obligate reseeder, is killed by fires and recruits with a pulse of seedlings. <i>Hypericum brachyphyllum</i>, a facultative reseeder/resprouter, both reseeds and resprouts after low intensity disturbances, but only reseeds after high intensity disturbances. <i>Hypericum brachyphyllum</i> and <i>H. chapmanii</i> are associated with intermediate, mesic areas along ecoclines where fires become increasingly heterogeneous to rare as they move down slopes. These species have low survival outside of their original habitats. Direct effects of fire, specifically removal of aboveground biomass and litter, increase seedling recruitment of <i>H. brachyphyllum</i> and <i>H. chapmanii</i>. Fires have no direct effect on the growth of <i>H. brachyphyllum</i> to juvenile and reproductive adult stages, but indirectly increase growth of <i>H. chapmanii</i>. This is likely the result of fire facilitating regeneration of associated nurse plants. Therefore, fires are important for seedling recruitment and growth of <i>Hypericum</i> species. Time to reproduction is longer for <i>H. chapmanii</i> (3+ years) and <i>H. brachyphyllum</i> (3 years) than <i>H. microsepalum</i> (2 years). Differences in time to reproduction should cause species to segregate along a fire frequency gradient. I thus conclude that heterogeneous fires within a high fire frequency landscape may increase persistence of <i>Hypericum</i> resprouters and reseeders in higher and lower areas along ecoclines, respectively. It should also allow plants with different life histories to co-occur in intermediate areas along ecoclines where fires are patchy and vary in intensity.

Pine savannas are an endangered ecosystem and cover <2% of their former range. Although often characterized by the presence of a single tree, P. palustris, the groundcover vegetation is extremely species rich. Thus, the groundcover vegetation is the focus of conservation efforts in pine savannas. This dissertation describes how fire, patches of shrubs, winter avian granivores, winter mammalian herbivores, and an introduced climbing fern (Lygodium japonicum) affect pine savanna groundcover. The fieldwork for this dissertation was done at Camp Whispering Pines, a pine savanna that has been undergoing restoration since 1990. Biennial, prescribed fires at the beginning of the growing season are a part of the habitat restoration. Although stem production of all groups of plants except bunchgrasses was stimulated by fire, annuals were found to respond most strongly to fire. Thus frequent fires appear to increase the presence of annuals in the groundcover of pine savannas. Pine savanna vegetation is interspersed with patches of shrubs. Along the margins of shrub patches there were more annual stems and fewer bunchgrass stems. Thus, shrubs appear to increase the heterogeneity of the groundcover. Pine savannas have a diverse group of avian granivores including some rare and endemic species. Avian granivores are probably selective in the seeds that they remove. Thus, they probably change the composition of the pool of seeds available to germinate. By removal of common species or large-seeded competitive dominants, avian granivores were predicted to increase the species richness of pine savanna groundcover vegetation. The White-tailed Deer (Odocoileus virginianus) and the Eastern Cottontail (Sylvilagus virginianus) are mammalian herbivores present at the study site. Mammalian herbivores often affect plant communities by removal of competitive dominants or selective removal of woody species. Mammalian herbivory was found to decrease the numbers of bunchgrasses, but did not affect other components of the plant community. In addition, bunchgrasses showed a large increase through the five seasons of data. There were, however, no subsequent changes as a result of this increase in stem numbers. Thus, bunchgrasses do not appear to competitively exclude other members of the species-rich groundcover community. Lygodium japonicum potentially threatens pine savanna groundcover. I evaluated how shrubs and fire affect its ability to invade pine savannas. Lygodium japonicum was found to occur in many more of the experimental plots along the margins of shrubs, and fire did not decrease its biomass. Presence of shrubs appears to facilitate invasion of pine savannas by L. japonicum, and fire is not an effective method of control.

The geographical range, abundance, and cohesion of populations can track landscape and climatic dynamism in ways that help set (and reset) the evolutionary trajectory of a species. Understanding the nature of this interaction can elucidate both evolutionary and geographical history as well as clarify the parameters that govern diversification. In this study, I apply multilocus genetic data from populations of the North American ground skink, Scincella lateralis, to investigate phylogeographic history as well as the interaction between population genetic and geographical processes that have shaped it. First, I examined the geographic distribution of genetic diversity for S. lateralis and tested whether an interaction between riverine barriers and climate-mediated population contractions have contributed to observed patterns. I found evidence for extensive mtDNA fragmentation resulting in 14 lineages and more moderate fragmentation of nuclear loci resulting in seven populations. The distribution and bounds of diversity are consistent with differentiation in response to riverine barriers that were rendered more isolating in the past when populations likely resided in southern refugia. I next applied multilocus sequence data and several analytical methods to reconstruct hierarchical relationships among S. lateralis populations and determine how dispersal has impacted divergence. In doing so, I explored the robustness of methods to assumption violations inherent when evaluating natural populations that have diversified recently and without complete isolation. Discordant hierarchical structure was recovered when using different methods and estimates of divergence and migration were invoked to evaluate population relationships. Rejection of a model of isolation-with-migration is largely contingent upon whether regions near adjacent populations are sampled, suggesting that recent gene flow following allopatric isolation is the primary mode of divergence. Finally, using mtDNA and microsatellite data I investigated the role of rivers in population genetics in more detail by estimating the current rate of S. lateralis migration across the Mississippi River and testing whether migration can be facilitated by meander loop cutoff, a common feature of major rivers. I found that gene flow across the river is high and largely asymmetrical in the direction predicted by cutoff-mediated dispersal, supporting one mechanism that may intermittently contribute to the permeability of riverine barriers.

Reef-dependent and reef-associated communities found on oil and gas platforms in the Gulf of Mexico occur in high densities and are species rich. Platforms may impact nearby communities through the introduction of benthic primary producers and by altering biological and environmental factors that may create influential gradients associated with these structures. To quantify the presence and effective distance exerted by such gradients, reef-dependent and reef-associated communities were analyzed in two ways: (1) community patterns of abundance and diversity were compared near and far from platforms; and (2) a food-web tracer was used to indentify basal resources from platforms used by consumers. Demersal and benthic communities, along with environmental gradients, were sampled 0.25 and 1.5 km from four operational platforms in the Gulf of Mexico. No distance effects were observed in demersal or benthic communities, metals concentration, organic carbon content of the sediment, or sediment particle size. A distance effect was detected in the abundance of Micropogonius undulatus (Atlantic croaker) and Centropristis philidelphica (rock sea bass). Prey depletion near platforms (i.e., a foraging halo) was not observed for potential demersal prey items of Lutjanus campechanus (red snapper) or in the benthic community. Reef-dependent communities at two platforms were sampled via SCUBA and analyzed using stable isotopes to determine the role of benthic algae from platforms in the diets of reef-dependent and reef-associated communities. Partial contributions of red algae endemic to platforms were observed in the crabs Pseudomedaeus agassizii, Teleophrys pococki, Micropanope nuttingi, stenothoid amphipods, and harpacticoid copepods as well as in the gut contents of L. campechanus and Balistes capriscus (grey triggerfish); however, phytoplankton is likely the dominant basal resource in both reef-associated and reef-dependent communities, contributing to over 78% of the diet of reef-dependent suspension-feeders. Dense suspension feeding communities may capture and process phytoplankton making secondary production and egestia available to reef-dependent and reef-associated consumers. No distance effects of stable isotopes were observed within reef-associated organisms or bulk sediment. Overall, the effects of platforms (both on community structure and food webs) are generally localized to < 250 m with species-specific effects on abundance, diversity, and diet extending to this distance.

Nopp140 is a nucleolar protein with several purported roles in ribosome biogenesis. To further characterize Nopp140 in Drosophila, we used FLP-FRT recombination to delete the Nopp140 gene. Genomic PCR, RT-PCR, and immuno-fluorescence microscopy confirmed the loss of Nopp140 and its products. Compared to embryos bearing the transposons used to generate the deletion, Nopp140-/- embryos displayed similar hatching rates, but resulting larvae died after 8 days, still in the second instar stage. Nucleoli were apparent in Nopp140-/- cells with no observable morphological defects, but the rRNA methyl-transferase, fibrillarin, redistributed partially to the nucleoplasm. BrU-labeling indicated that rDNA transcription is reduced overall in Nopp140-/- larvae, while Northern analysis showed that pre-RNA cleavage was un-affected. Surprisingly, Northern analyses of Nopp140-/- larvae showed an unusually large pre-rRNA bearing the R2 retro-transposon sequence and the processed R2 transcript. The observation suggests a fundamental shift in rDNA chromatin structure and expression upon loss of Nopp140. Transmission electron microscopy of Nopp140-/- cells showed excess nuclear virus-like copia particles, reduced cytoplasmic ribosomes, autophagosome-like structures, and many electron dense cytoplasmic granules that are likely to be stress bodies and proccessing (P) bodies. Because the downstream piggyBac element used to delete Nopp140 resides in CG7145, phenotypes caused by this insertion were compared to those caused by Nopp140 deletion. Delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) is a nuclear-encoded mitochondrial enzyme that catalyzes the second step in proline degradation. Mutations in human P5CDh cause type II hyperprolinemia, a complex syndrome displaying increased serum proline and mental disabilities. Conceptual gene CG7145 in Drosophila melanogaster encodes the orthologous DmP5CDh1. The mutant allele, CG7145f04633, contains a piggyBac transposon that truncates the enzyme by 83 residues. Heterozygous (CG7145f04633/TM3) individuals developed normally, while homozygous (CG7145f04633/CG7145f04633) individuals displayed proline levels twice that of normal, swollen mitochondria, and ultimately larval and pupal lethality. These mutants showed normal ribosomal enrichment in cytoplasm, and normal rRNA gene transcription, processing, and pre-rRNA modifications. We conclude that the Nopp140 plays a critical role in ribosomal biosynthesis, and that the phenotypes in Nopp140-/- can be distinguished from the phenotypes caused solely by the CG7145 mutation.

Forest regeneration on abandoned land in the Brazilian Amazon depends first and foremost on prior land-use history. Abandoned clearcuts become dominated by Cecropia trees, but contain a rich mix of other arboreal genera and succession proceeds rapidly to a diverse forest. In contrast, abandoned land that has been burned repeatedly for pastures becomes dominated almost completely by Vismia trees and remains in monogeneric stands gaining few new genera over the same time interval. I tested the hypothesis that areas with repeated burns would have more re-sprouts, particularly Vismia re-sprouts. I also predicted that in monogeneric Vismia stands, seed dispersal would be limited to few bat-dispersed genera while there would be more diverse seed rain in Cecropia, particularly from bird dispersers. In order to test these hypotheses, stands with different land-use histories were evaluated in terms of tree, palm, shrub and liana abundance and diversity, as well as whether they germinated from seed or grew as re-sprouts from the root. I found that Vismia stems were one hundred percent re-sprouts in Vismia stands. In Cecropia stands, 93% of Vismia stems germinated from seed. There were no significant differences in the abundance of re-sprouts by Cecropia and all other species, regardless of stand type. To test how seed dispersal in Cecropia and Vismia stands differs, I collected seeds fallen into seed traps by bird and bat dispersers as well as from fecal samples collected principally from mist netted birds. I found that both bird and bat dispersers deposited a more diverse seed assemblage into Vismia stands than Cecropia stands. Overall, bats disperse more Vismia seeds while birds disperse more Miconia seeds, regardless of stand type. My results suggest that seed dispersal does not drive the differences in succession found in Cecropia and Vismia second growth. However, the capability of seeds to germinate and recruit in Vismia stands warrants further investigation. I suggest that the use of repeated fire should be limited as much as possible due to the long term effects on succession after site abandonment.

DNA damage is a common hazard that all cells have to combat. Saccharomyces cerevisiae HMO2 is a high mobility group protein (HMGB) that is a component of the chromatin remodeling complex INO80, which is involved in double strand break repair. I show here using DNA end-joining and exonuclease protection assays that HMO2 binds preferentially to DNA ends. While HMO2 binds DNA with both blunt and cohesive ends, the sequence of a single stranded overhang significantly affects binding, supporting the conclusion that HMO2 recognizes features at DNA ends. Analysis of the effect of duplex length on the ability of HMO2 to protect DNA from exonucleolytic cleavage suggests that more than one HMO2 must assemble at each DNA end. HMO2 binds supercoiled DNA with higher affinity than linear DNA and has a preference for DNA with lesions such as pairs of tandem mismatches; however, comparison of DNA constructs of increasing length suggests that HMO2 may not bind stably as a monomer to distorted DNA. The remarkable ability of HMO2 to protect DNA from exonucleolytic cleavage, combined with reports that HMO2 arrives early at DNA double strand breaks, suggests that HMO2 may play a role in double strand break repair beyond INO80 recruitment. I also found that HMO2 has the ability to mediate both 3 and 5 DNA strand invasion, which is an essential step in homologous recombination. Also hmo2∆ and hmo2∆rad52∆ have slower growth phenotype in presence of hydroxyurea thus indicating that HMO2 might play important role in recovery of stalled DNA replication forks.

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled and expressed heterologously in Streptomyces lividans. Bioinformatic analysis and mutagenesis studies elucidated the biosynthetic pathway at the genetic and biochemical level. Four gene sets, asuA-D, govern the formation and assembly of the asukamycin building blocks, a 3-amino-4-hydroxybenzoic acid (3,4-AHBA) core component, a cyclohexane ring, two triene polyketide chains and a 2-amino-3-hydroxycyclopent-2-enone (C5N) moiety to form the intermediate protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of protoasukamycin to 4-hydroxyprotoasukamycin, which is epoxidized at C5-C6 by AsuE3 to the final product, asukamycin. Branched acyl CoA starter units, derived from Val, Leu and Ile, can be incorporated by the actions of the polyketide synthase KSIII AsuC3/C4 as well as the cellular fatty acid synthase FabH to produce the asukamycin congeners A2-A7. In addition, the type II thioesterase AsuC15 limits the cellular level of ù-cyclohexyl fatty acids and likely maintains homeostasis of the cellular membrane.