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.

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.