Recessive mutations in the ENS1 gene of Arabidopsis thaliana result in reduced trichome branching without altering the ploidy level of trichomes. This implies that ENS1 regulates trichome branching in an endoreplication-independent manner. Mutations in ENS1 also enhance the multicellularity of sim mutant trichomes indicating that ENS1 plays a role in the regulation of the cell cycle during trichome cell differentiation. We have shown that ENS1 is required by the negative regulators of trichome branching for the trichomes to achieve their supernumerary branching. The interaction between ens1-1 and try-JC suggests that ENS1 is involved in both primary and secondary branching events during trichome development. The ENS1 protein interacts both genetically and physically with the STI protein and double mutant analysis between ens1-1 and sti mutants revealed that they function in the same pathway during trichome development. We propose that ENS1 and STI form a complex that functions to ensure the development of a wild-type trichome phenotype. The ens2-1 mutations result in trichomes with reduced branching and reduced endoreplication. The ens2-1 mutations also enhance the multicellularity of sim mutant trichomes. The ENS2 gene encodes an activator of the APC/C, a multisubunit protein complex that targets proteins for degradation by the 26S proteasome. Overexpression of ENS2 and its close Arabidopsis homologue AtCCS52A2 results in plants with retarded growth that have enlarged leaf epidermal pavement cells containing highly endoreplicated nuclei. These plants also have large multibranched trichomes with highly endoreplicated nuclei. Both ENS2 and AtCCS52A2 suppress the sim mutant trichome phenotype, suggesting these two genes are functionally similar and function in the same pathway in the regulation of the endoreplication cell cycle. The ens2-1 mutation induces multicellular trichomes in plants overexpressing cell cycle regulators cyclin B1;1, cyclin B1;2, cyclin D4;1 and CDKA;1, indicating cyclin D4;1 and CDKA;1 may be involved in the regulation of the G2/M as well as the G1/S phase of the cell cycle. Our research findings indicate that ENS1 and ENS2 are involved in the regulation of the cell cycle during trichome development and that the cell cycle and the cell shape mechanism interact during trichome development to ensure the development of the wild-type trichome architecture.

For nearly two centuries, it has been known that biological diversity is not homogeneously distributed across the planet, and yet a general explanation for this variation remains elusive. Hundreds of studies have found that taxonomic diversity (i.e., richness) is strongly associated with characteristics of the environment. Measures of energy or climate seem to be particularly strong correlates of richness, while measures of environmental heterogeneity are typically of secondary importance. The pervasiveness of richness-environment correlations has been seen as evidence that diversity gradients result from environmental gradients. However, effects of environment on diversity may have been overestimated. In this dissertation, I used comparative analyses and computer simulations to test whether environmental energy and climate are truly general determinants of richness gradients at broad scales. I found that energy and climate are strong correlates only of diversity patterns of species with broad distributions. Richness of narrowly distributed species is only weakly associated with environment, and it is heterogeneity (not energy or climate) that better accounts for richness of these species. This questions the idea that environment (particularly energy/climate) is an important and general determinant of diversity gradients, as richness of a large proportion of species (those with narrow distributions) are not strongly associated with environmental characteristics. Moreover, I found that the stochastic diversification of clades can produce frequent, but spurious, richness-environment relationships. Thus, pervasiveness of empirical richness-environment correlations might not be reliable evidence for environmental effects on the production of richness gradients. This also suggests that re-evaluation of richness-environment relationships using appropriate null models, which incorporate the diversification process, could lead to important new insights about the determinants of richness patterns. For example, I found that when comparing empirical richness-environment relationships to those expected by random clade diversification, the perceived effects of energy/climate are reduced while the effect of environmental heterogeneity is increased. This suggests that heterogeneity might play a more significant role in the formation of richness gradients than previously assumed. My results have important implications for theories developed to explain diversity gradients, and for efforts to predict the future of biodiversity in the face of large scale changes in climate.

Herbivorous insects are known to negatively impact plant fitness, such that plants have evolved defense strategies to reduce the preference and performance of herbivores on those plants. However, a plants investment in defense may be costly when herbivores are absent from the environment. Defense traits that are induced only upon herbivory can mitigate costs associated with defense maintenance. Although costs and benefits of induced responses are generally assumed, empirical evidence for them is equivocal. We examined the fitness costs and benefits of jasmonic acid-induced responses by soybean (Glycine max L. Merr.) in the absence and presence of soybean loopers (Chrysodeix includens Walker) (Lepidoptera: Noctuidae), an economically important pest of soybeans in the southern United States. In a greenhouse experiment we demonstrated that induction was costly to soybeans, affecting all components of soybean fitness. Jasmonic acid-induced plants produced 10.1% fewer seeds that were 9.0% lighter, and had 19.2% lower germination rates than control plants. In contrast, induction provided only modest benefits to soybeans. In a choice experiment, soybean loopers exhibited a significantly greater preference for leaves from control plants consuming 62% more tissue than from jasmonic acid- induced plants. Soybean loopers that did feed on induced soybean plants matured at the same rate and to the same size as soybean loopers that fed on control plants. However, at high conspecific density, soybean looper survivorship was reduced by 44% on induced relative to control plants. Negative effects of induction on soybean looper preference and survivorship did not translate into fitness benefits for soybeans. Our study is the first evaluation of costs and benefits of soybean induced responses.

Multiple classes of retinal amacrine cells use L-type Ca2+ channels to mediate synaptic transmission. I have used whole cell voltage clamp recordings from primary cultures of amacrine cells to investigate the regulation of these channels. In this study, I show that inhibiting mitochondrial calcium uptake (MCU) caused a reversible reduction in the Ca2+ current amplitude. Replacing external Ca2+ with Ba2+ minimized the effects of blocking MCU indicating that the Ca2+ influx is the primary source of the inhibition. With 1,2-bis-(o-aminophenonxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) in the recording pipette, MCU inhibition caused an increase in the current amplitude indicating that the fast buffering capability of BAPTA minimizing the Ca2+-dependent inactivation of the channels, revealing a Ca2+-dependent enhancement, possibly through protein kinase A (PKA) activity. The effect of a PKA inhibitor was consistent with this possibility. Inhibiting the calcium-induced calcium release (CICR) also decreased the Ca2+ current amplitude. These results indicate that MCU, PKA and CICR are critical to maintain the availability of L-type Ca2+ channels for depolarization-dependent signaling in amacrine cells. Production of nitric oxide (NO) by neuronal and endothelial nitric oxide synthases (nNOS; eNOS) is another Ca2+-dependent mechanism in the retina. In the chicken retina, I demonstrate the cell-autonomous nature of the NO signal by comparing the pattern of NO production to the expression of NOS. The NO indicator fluorescence dye, DAF was used to detect the pattern of NO production. A NOS inhibitor L-NAME suppressed the DAF signal suggesting that the source of DAF-signal was due to NOS activity. I also demonstrate the presence of NOS-immunoreactivity in the chicken retina. Neuronal NOS and eNOS antibodies labeled photoreceptors, amacrine cells and cells in the ganglion cell layer (GCL). Anti-e NOS also labeled horizontal cells, a small subset of bipolar cells and Müller cells. Different subsets of amacrine cells were labeled in dorsal and ventral retina with anti-nNOS. Endothelial NOS labeling did not show difference in dorsal and ventral retina but expression was more wide spread than nNOS. These results suggest that the potential for NO production is wide spread in the avian retina.

Vibrio vulnificus is a gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by the ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype (OpS), while unencapsulated attenuated strains appear translucent (TrS). A third colony type, rugose (R), is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. V. vulnificus undergoes spontaneous phase variation associated with altered levels of CPS and rEPS, and the work presented here identifies genetic and environmental parameters that control this process. A cluster of nine genes (brpABCDFHIJK) was found to be up-regulated in R isolates when compared to OpS or TrS isolates. We assessed the role of the brp gene cluster in CPS and rEPS production by creating non-polar mutants and characterizing their colony morphotypes, rEPS production, biofilm formation, and motility phenotypes. We demonstrate that the brp genes are required for rEPS production and robust biofilm formation, and that the decreased motility of R isolates is subject to regulation by the second messenger cyclic-di-GMP. Approximately 130-135bp upstream of brpA, we identify a promoter, which is activated at significantly higher levels in R variants than in TrS or OpS variants. In addition to characterizing the genetic components of rEPS phase variation, we show that environmental factors, such as media composition and temperature, influence the rate of polysaccharide phase variation. Specifically, calcium (Ca2+) significantly increases the rate of CPS and rEPS phase variation in V. vulnificus. Multiple phenotypic responses to increased [Ca2+] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain TrS isolates contained deletions at the group I CPS operon, inferring increased [Ca2+] up-regulates existing phase variation mechanisms. Expanding on a previous observation, increased [Ca2+] also enhanced biofilm formation for all phase variants. Our results show that [Ca2+] increases polysaccharide phase variation and contributes to biofilm formation, thereby likely playing a dual role in the persistence of V. vulnificus in the environment.

Boundary elements consisting of barriers and insulators are genomic sequence elements that along with their associated DNA-binding proteins block the spread of heterochromatin into euchromatic regions or prevent the targeted activation of promoters from distal/proximal enhancers, respectively. In Saccharomyces cerevisiae, the deletion of RPD3, a histone deacetylase, results in an extended SIR protein-mediated silencing effect bypassing a tRNAthr barrier element adjacent to the cryptic mating locus, HMRa. We mutagenized rpd3Δ strains and identified suppressor mutants through a genetic screen that no longer displayed this enhanced silencing effect. Our results identified BRE1 and BRE2, which are either directly or indirectly responsible for the tri-methylation of histone H3K4 and H3K79, as effectors of the rpd3Δ extended silencing effect at HMRa. We hypothesize that the increased silencing effect in rpd3Δ mutants is the result of a redistribution of SIR proteins which become concentrated at the HMRa region in response to a global change in the acetylation and/or methylation state of histones contingent on RPD3, BRE1, and BRE2. ETC, or Extra-TFIIIC, sites are genomic elements which bind the RNA Polymerase III transcription factor, TFIIIC. ETC sites contain B-box promoter sequences normally associated with RNA Polymerase III promoters, and their locations are over-represented between divergently transcribed RNA Polymerase II genes. Our results show that the transcription of TFC6, which codes for a DNA-binding component of TFIIIC, is auto-regulated by TFIIIC which binds to the ETC6 site in the TFC6 promoter region. Inhibition of TFIIIC binding to the ETC6 site results in increased TFC6 expression from its own promoter, and transcription of TFC6 is inversely correlated with TFIIIC binding to the ETC6 site. The TFC6 promoter is also down-regulated when its own gene product is over-expressed. We present here a novel function of gene regulation where a Pol III transcription factor directly (auto) regulates a Pol II gene. Our results also point to how this regulation might be mediated by an insulator-like function of TFIIIC which can implicate the functionality of Extra-TFIIIC sites in other eukaryotes.

Acetyl-CoA carboxylase is an essential enzyme, as it catalyzes the first committed and regulated step in fatty-acid biosynthesis in all organisms excepting few Archaea and Eubacteria. Acetyl-CoA carboxylase from gram-negative and gram-positive bacteria is a multifunctional enzyme composed of three separate proteins. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from carboxybiotin to acetyl-CoA, forming malonyl-CoA. The crystal structure of the Escherichia coli (E. coli) carboxyltransferase component of acetyl-CoA carboxylase revealed a unique Zn-domain, presumed to mediate nucleic acid binding, that is absent in the eukaryotic enzyme. Notably, the Zn-domain, adjacent to the active site of carboxyltransferase, makes for a unique target in the development of novel antibiotics capable of highly specific binding. Utilizing an Electrophoretic Mobility Shift Assay as part of this study, we investigated the nonspecific nucleic-acid binding and substrate (malonyl-CoA and biocytin) inhibition of DNA:carboxyltransferase complex formation. Inhibition of carboxyltransferase activity by single-stranded DNA, double-stranded DNA, RNA, and heparin was measured in the reverse direction with a spectrophotometric assay in which the production of acetyl-CoA was coupled with the combined citrate synthase-malate dehydrogenase reaction requiring NAD+ reduction (Blanchard and Waldrop, 1998). NADH formation was followed spectrophotometrically at 340 nm. We then determined and characterized the mechanism of inhibition by tetracycline (and derivatives) on carboxyltransferase from E. coli and Staphylococcus aureus. The tetracyclines are broad-spectrum antibiotics that inhibit translation by binding to the 30S ribosomal subunit and preventing the binding of the acylated-tRNA to the A-site. Tetracycline exhibited competitive inhibition with respect to both malonyl-CoA and biocytin. Multiple inhibition analyses with a bisubstrate analog showed that tetracycline and the substrates can bind to the enzyme simultaneously. Surprisingly, tetracycline did not interfere with the DNA-binding properties of carboxyltransferase. This introduction begins with a historical perspective of carboxylation reactions. Next biotin and the structure, function and practical applications of acetyl-CoA carboxylase are described. Subsequently a review of moonlighting enzymes, or those capable of catalyzing reactions in basic metabolism while acting as regulators of gene expression, is provided, as are the functions and structures of several types of zinc finger.

Mummichog (Fundulus heteroclitus) responses to chronic whole-ecosystem nutrient enrichment were examined near Plum Island Sound, Massachusetts. Dissolved fertilizer was released into replicate salt marsh creeks over 6 growing seasons to simulate agricultural run-off (bottom-up effects). Density, biomass, secondary production, growth rate, and condition factor were estimated in fertilized and reference creeks. Mummichog densities were also used to determine if mummichog growth or health varied with density. Over 7,600 mummichogs were marked and released into the treatment and control areas to measure responses. Over 900 mummichogs were recovered. Mummichog abundance was higher (p = 0.055) in nutrient-enriched creeks than reference creeks (0.81 ± 0.04 fish m-2 and 0.59 ± 0.07 fish m-2 respectively). Nutrient enriched-creek biomass of 522.9 ± 36.1 mg dw m-2 was significantly higher (p=0.028) than control-creek biomass of 338.5 ± 26.7 mg dw m-2. However, reference-creek growth rates of 0.105 ± 0.091 were significantly higher (p=0.04) than the nutrient enriched-creek growth rates of 0.073 ± 0.065 mm d-1. Secondary production and condition factor of mummichogs did not differ with nutrient enrichment. Nutrient enrichment likely stimulated primary production causing bottom-up effects in the food web, which increased mummichog abundance and biomass. However, as abundance increased, mummichog growth rates decreased, suggesting a density-dependent response, likely caused by either intraspecific competition or behavioral changes causing dietary shifts.

Most biogeographic studies on the Mexican biota have assumed that the dramatic climate cycles of the Pleistocene epoch and the prominence of the Trans-Mexico Volcanic Belt have played major roles in the origin and diversification of species. Here the pylogenetics and biogeography of four codistributed rodent species were studied. In each case, a phylogenetic hypothesis for the taxon and allied species using two mitochondrial (Cytochrome-b and 12S), and two nuclear genes (GHR and IRBP) was generated, appropriate taxonomic changes were recommended, and a temporal framework was generated to identify events that may have produced the phylogenetic pattern. Nelsons woodrat Neotoma nelsoni and the Perote ground squirrel Xerospermophilus perotensis, were confirmed as having their closest relatives in the Mexican Plateau. The findings also confirmed that N. nelsoni and X. perotensis are genetically well-differentiated from their sister taxa. Genetic distances in combination with low levels of morphological differentiation suggest that they should be recognized only at the subspecific level as N. leucodon nelsoni and X. spilosoma perotensis. Molecular estimates of divergence times suggested that N. l. nelsoni and X. s. perotensis diverged from their sister taxa to the north during early Pleistocene times. The rock mouse Peromyscus difficilis was divided into two well-supported clades, a northern clade including the subspecies P. d. difficilis and P. d. petricola, and a southern clade containing the subspecies amplus, felipensis, and saxicola. Molecular-based estimates of divergence times suggested that separation of these clades occurred in the Pleistocene. The study of the Phillips kangaroo rat, Dipodomys phillipsii, revealed a biogeographic pattern different from that seen for other taxa. D. phillipsii was divided into two well-supported clades: one distributed on the Mexican Plateau, and a southern clade in the TMVB. Several lines of evidence supported the decision to return the Mexican Plateau clade of D. phillipsii to full species status as D. ornatus. The study showed that D. phillipsii, D. ornatus, D. elator, and D. merriami form a well-supported clade of kangaroo rats, however, the dataset was unable to resolve relationships among these four species. Molecular-based analyses of divergence times suggests that D. phillipsii, D. ornatus, D. elator, and D. merriami diverged in mid-Pliocene times, probably in or near the Mexican Plateau. Unlike the Pleistocene divergence dates reported in previous chapters this Pliocence divergence suggests that the morphotectonic processes that gave rise to the Trans-Mexico Volcanic Belt may have influenced early diversification in Mexican species of Dipodomys.

Research in invasion ecology has focused on developing ecological theory that can predict how invasive species interact with invaded communities. However, empirical support for theoretical predictions has been inconsistent. Inconsistencies may be attributed to the lack of data in three core areas; (1) field data with enough resolution to determine population dynamics of invasive species in relation to native species, (2) manipulative field experiments that encapsulate natural variation found among micro-habitats, and (3) field data that incorporate effects of invasive species within and among spatial scales. This dissertation has addressed these concerns by examining the mechanisms responsible for the successful invasion and ecological impacts of the invasive grass smooth brome (Bromus inermis) within the prairies of North Dakota and Minnesota. GIS analyses revealed that native Spartina pectinata patch growth was two times greater in non-invaded areas versus areas heavily invaded with brome. The probability of extinction of native Spartina pectinata averaged 8 times more likely in areas of high versus low brome coverage. Field experiments determined differences in germination between invasive smooth brome and native prairie cordgrass were not driven by habitat differences or soil conditions. Following initial germination, invasive smooth brome had a negative impact on cordgrass establishment, which was primarily due to a 78% and 47% reduction in native cordgrass plant height and stems density, respectively. Throughout the field experiment invasive smooth brome was a dominant competitor under all habitats and soil conditions except in areas where soil salinity levels were highest. Results from the herbivore study indicated that smooth brome has the potential to have negative effects on local herbivore assemblages. Despite the large differences in herbivore species richness, diversity and evenness at our larger spatial scales, my results indicated no statistically significant effects of invasive smooth brome. Results indicated that plant species richness had a larger effect than invasive smooth brome on herbivore assemblages (i.e. plant species richness, predator abundances, landscape surroundings). Future directions for research concerning the impact of smooth brome on native herbivore assemblages should include incorporating herbivore community composition, predator abundances and landscape features (i.e. surrounding matrix, prairie isolation and management history).