The sale of small turtles is banned from the US market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses 1000 μg/ml gentamicin sulfate baths to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria such as Salmonella enterica subsp. enterica, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2000 μg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) contributing to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate their incidence in these bacteria, as well as to explore the molecular elements that contribute to the dissemination of gentamicin resistance in this context. R-plasmids from gentamicin resistant strains were transferred by conjugation and transformation to naïve Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of aminoglycoside acetyltransferase genes aac(3)IIa and aac(3)VIa. Multiplex PCR assays showed that every gentamicin resistant isolate carried either one of these acetyltransferase genes. Pulsed-Field Gel Electrophoresis and restriction enzyme digestion analysis of R-plasmids carrying these genes revealed plasmids with sizes ranging from ca. 45 kb to 145 kb, with different restriction profiles, supporting their presence on mobile molecular elements. In effect, gene aac(3)VIa was present as a gene cassette of a class 1 integron. Furthermore, evidence suggests that gene aac(3)IIa may be encoded as part of a complex class 1 integron. The presence of aac(3)IIa and aac(3)VIa in mobile molecular elements explains the distribution of these genes in the different plasmids and organisms described in this study. Further studies are required to characterize these molecular elements, and to detect genes which may be co-selected in the presence of gentamicin. This work is one of the first to report dissemination of high-level gentamicin resistance in Gram-negative bacteria, particularly in environmental isolates of Salmonella enterica subsp. arizonae.

Arabidopsis thaliana is an organism that can be used as a model for most of the processes that occur in flowering plants. The leaf hairs, or trichomes, of Arabidopsis thaliana are macroscopic single cells that have been used as a model system for cell fate determination, cell expansion, cell cycle regulation, cell wall deposition, as well as other processes. Initiation of the trichome cell fate is controlled by a complex of genes including GLABRA1 (GL1), TRANSPARENT TESTA GLABRA (TTG), and GLABRA3 (GL3). This work examines the role of GL3 in trichome initiation and uses plants expressing varying levels of GL3 to determine if genes involved in trichome development are regulated by GL3. Though several genes are given a cursory examination, the regulation of two genes, an α-carbonic anhydrase and a novel cell-cycle regulator called SIAMESE, are given a thorough examination. The α-carbonic anhydrase At2g28210 was previously not known to be involved in trichome development. Its involvement in trichome development was discovered with the aid of an enhancer trap line with robust reporter gene expression in developing trichomes. Pharmacological studies indicate that this α-carbonic anhydrase may play a role in trichome expansion. SIAMESE (SIM) was first identified in a mutant screen in the Larkin lab. This dissertation demonstrates that this gene encodes a novel type of cell-cycle regulator with several homologs in Arabidopsis and other plant species. SIM and one of its homologs in Arabidopsis were shown to be expressed in a trichome-dependent manner. These investigations shed new light into the molecular process of trichome development.

Vibrio vulnificus is a marine bacterium capable of causing severe and fatal illness in susceptible people. The primary virulence factor is a capsular polysaccharide (CPS) that allows the bacteria to evade the host immune system. Two phenotypic forms were previously known to exist, opaque (virulent) and translucent (avirulent), and had been shown to undergo spontaneous phase variation, each switching to the other phenotype. Provided here is the first report of a third phenotype, rugose. The rugose variant is dry and wrinkled compared to the smooth colony morphology of the opaque and translucent forms. Both opaque and translucent strains produce rugose variants that are similar in some ways, but show important differences between the two. Rugose V. vulnificus produce copious amounts of rugose exopolysaccharide (EPS) that allows them to form well-defined biofilms in attachment to glass, as a pellicle on the surface of broth, and as a 3-dimensional colony of distinct architecture. The rugose forms survived better in normal human serum than did their opaque and translucent parents, while only the translucent parent and its rugose variant survived exposure to chlorine. It is proposed that opaque-derived rugose variants also produce CPS, in addition to EPS, and that it is the CPS that actually confers susceptibility to chlorine. Five genes associated with CPS and/or EPS production were expressed by all phase variants at all points of the life cycle. Also reported here is a group of genes, the rugose gene cluster, differentially expressed by the three phenotypes. Opaque and translucent forms expressed genes in this cluster at low levels or not at all, while the rugose variants expressed all of these genes at higher levels throughout exponential growth. As biofilms have been shown to protect bacteria from adverse environmental conditions and the rugose variants produce robust biofilms, this form may contribute to the survival of this bacteria in its natural environment. Future study of these genes should provide insight into the expression and regulation of the rugose phenotype and the resulting biofilms, as well as survival and virulence of V. vulnificus.

The Southeastern United States has the most diverse and imperiled freshwater mussel (Unionidae) fauna in the world. The community structure and decline of these organisms is the result of complex interactions between biotic and abiotic factors, but the limited spatial scale of most community studies has failed to elucidate the underlying mechanisms shaping community structure. Basing community assessments solely on microhabitat variables alone has lead to stark contradictions in management recommendations and opposing definitions of habitat requirements for these organisms. However, with the introduction of GIS technology into aquatic management, it is now feasible to include variables from larger spatial scales and investigate previously undetectable mechanisms influencing unionid community structure. In my study, I tested the hypothesis that patterns of mussel species richness and abundance in the Tickfaw, Tangipahoa Bogue Chitto and West Pearl rivers in south-eastern Louisiana were related to a combination of local-scale habitat variables, riparian-scale land use and geology variables, and sub-segment scale land use and geology variables. ANOVA results indicated a significant difference between the upper three sub-segments and lower three sub-segments of these rivers. The Principal Component Factor Analysis (PCFA) results revealed that geology, land-use and fine sediment are components working together across spatial scales to produce a hydrologic variability mechanism, and a simple regression model based on the factor scores of each site was successful in predicting abundance and species richness. Through comparing the ANOVA results with the factor score results for each site, I conclude that hydrologic variability, defined by geology type and land-use as well as fine sediment, is influencing the pattern in freshwater mussel abundance and species richness found in the Florida Parishes of Louisiana.

Effects of hydrocarbons, salinity, tidal height, bioenergetics and competition on the oyster reef fauna were studied. Dried oyster shell was exposed to crude oil in the laboratory and plastic trays filled with control and oil-exposed shell were then deployed at two locations, in two seasons and at two tidal levels, with immersion periods of two and five weeks. Salinity exhibited a significant effect with sub tidal site having higher diversity. Hydrocarbon effects were less prominent. To determine effects of cleaners, Corexit 9580 was applied both alone and in combination with oil on cultch. At high concentrations, the cleaner ameliorated hydrocarbon effects. Hydrocarbon effects seemed to be less prominent than salinity and aerial exposure. In the next chapter, effects of salinity on the survival and bioenergetics of mud crabs Panopeus simpsoni and Eurypanopeus depressus were examined. Crabs were exposed to a range of salinities to determine the effects of salinity on tolerance. P. simpsoni exhibited a 28d LC50 of 6.97 PSU while E. depressus had a 28d LC50 of 0.19 PSU. Crabs were exposed to four salinities for bioenergetic measurements. Energy expenditure was highest at the lowest salinity and decreased as salinity increased. Scope for growth declined below 17.5 PSU. E. depressus was capable of surviving lower salinities than P. simpsoni. However, the physiological responses do not significantly differ between the two species. In the next chapter laboratory experiments were conducted to see if these two crab species differ in resource holding potential. Crabs were placed in aquaria at two salinities and refugia were checked daily for shelter occupancy. To determine if resource holding potential for refugia influenced predation risk, a second laboratory experiment was performed with a blue crab predator in each tank. The numbers of mud crabs of each species surviving were recorded. E. depressus was dominant over P. simpsoni in occupying the shelters at both salinity levels. E. depressus exhibited a higher survival in the presence of the predator. E. depressuss ability to tolerate lower salinities, and its dominance in resource holding potential, may lessen predation risk and allow colonization of more estuarine sites.

Analyses of ITS, ETS and matK sequences for genera of the subtribe Hinterhuberinae, including representatives of 17 subtribes from the tribe Astereae, rooted on Anthemideae, Calenduleae and Inuleae, provide no support for the monophyly of the Hinterhuberinae. Chiliotrichum and Celmisia groups, Olearia in part, Novenia, Oritrophium and Pteronia are in the basal group that also includes subtribe Felicinae. Madagaster, Remya, Olearia in part, Achnophora and Hinterhubera group lie in the Southern Hemisphere grade, with Asterinae, Bellidinae, Brachycominae, Grangeinae, Podocominae, Lagenophorinae and Baccharidinae. Monophyly of Asterinae, Grangeinae, Podocominae and Lagenophorinae is not supported. Instead South American representatives of Lagenophorinae, Grangeinae and Podocominae group with Baccharidinae and Hinterhubera group in the South American clade that is nested in the Southern Hemisphere grade. They are sister to the North American subtribes. Remya, Achnophora, Olearia in part and Australasian, European and African representatives of Asterinae, Brachycominae, Grangeinae, Podocominae and Lagenophorinae are grouped in a polytomy that is basal to the South American clade. Laennecia (Podocominae) is sister to Westoniella (Hinterhuberinae), and Myriactis panamensis (Lagenophorinae) and Plagiocheilus (Grangeinae) group with Archibaccharis (Baccharidinae). The Celmisia group is monophyletic. The radiate Diplostephium and Guynesomia do not group with other radiate genera from the Chiliotrichum group, but are in the Southern Hemisphere grade and close to the disciform Hinterhubera group. Transfer of Blakiella from Podocominae to Hinterhuberinae is supported by the three datasets. Madagaster is isolated from the other Hinterhuberinae in having basal position in the Southern Hemisphere grade. Evolution of dioecy in Baccharidinae and Aztecaster (Hinterhuberinae) evolved independently. Woody habit, epaleate receptacle, bisexual disk florets and ligulate peripheral florets are ancestral states in the tribe Astereae. Staminate disk florets and paleate receptacles are derived characters that evolved in several lineages independently. South America, southern Africa or New Zealand are proposed centers of origin for Astereae and for the Chiliotrichum and Celmisia groups. The Hinterhubera group evolved from the South American autochthonous elements, probably from the lower altitude plants during the uplift of the Andes and the development of páramo habitat.

Coevolution between amino acid residues and its context-dependence are important for exploring protein structure and function, and critical for understanding protein structural and functional evolution. Coevolution has long been ignored because of its complexity and the lack of computing power. In the research presented here, I developed an efficient coevolution analysis methodology based on likelihood comparisons of statistical models. Likelihood ratios and Bayes factors, calculated using the Markov chain Monte Carlo algorithm, were employed as the statistics. Two types of models, 2-state and 3-state, were developed to allow for the context-dependence of coevolution. Computer programs implementing this methodology were coded in C/C++ and were run on the Beowulf clusters of our laboratory and the super computers of LSU. Using these programs and custom Perl scripts, residue coevolution in cytochrome c oxidase and photolyases/cryptochromes protein superfamily was analyzed. I found that pairwise coevolution between residues is highly dependent on protein tertiary structures and functions. I detected extensive coevolving pairs in all our analyses, and these pairs were primary localized in regions of known structural and/or functional importance. I also found that coevolution is related to evolutionary rate and concentrated in moderately conserved sites. In supporting the importance of functional constraints, I detected a non-negligible coevolutionary signal between complex subunits and stronger coevolution in proteins of functional importance. I also found that the interaction between subunits can serve as a local coevolutionary constraint on one subunit rather than driving coevolution between two subunits. Based on coevolutionary patterns, I suggested that a domain without any previously supposed function actually operates as a folding core in the proteins of photolyase/cryptochrome superfamily. The coevolutionary patterns also provided clues regarding the functional evolution of electron transfer in this superfamily. I also found that coevolving sites with double substitutions along a branch tend to occur only at physically contacting sites, and that salt-bridge stabilization and secondary structure stabilization are important forces of residue coevolution. The methodology and programs developed in this research are powerful tools for coevolutionary analysis, which can provide valuable information for characterization of protein structural/functional domains and exploration of protein structural/functional evolution.

Recently, much evidence has been brought forth into the scientific community supporting the idea that RNA Polymerase III transcribed regions of DNA may serve as chromosomal landmarks for silencing. Transfer RNA genes are known to involve themselves in several extra-transcriptional functions within the chromosome, including the pausing of replication forks, Ty element integration, tRNA position effects (repression of neighboring genes), acting as a barrier to the spread of heterochromatin, and over-riding nuclosome positioning sequences. Our results suggest that many tRNA genes may serve these functions as well as exhibiting behavior similar to metazoan insulators. Also, ETC (Extra TFIIIC) sites within Saccharomyces cerevisiae, which bind only TFIIIC may also act as barriers or insulators. Our results support the idea that extra-transcriptional functions of RNA Polymerase III factors may be widespread and important contributors to genome biology.

Epigenetic modification is another mechanism involved in the cancer development besides classic mutations such as deletion. Aberrant promoter methylation and associated chromatin modification have been frequently reported in various tumors of different clinical stages. Hypermethylation has been frequently observed in tumor suppressor genes and causes reduced transcripts of these genes. Hypomethylation also has been reported to be involved in activation of oncogenes. Inactivation of the X chromosome and the imprinting of gamete DNA depend on the methylation patterns in the promoter region. Understanding methylation mechanisms could be helpful to diagnosis of early stage tumorgenesis or offer molecular markers for detecting cancers. Changes in methylation patterns based on human vulva pathological tissue type of four genes have been studied in the current project. RASSF1A and DAPK-1 are tumor suppressor genes. BRCA2 is considered highly associated with breast and ovarian cancer. And H19 is a maternal imprinted gene. DAPK-1 and BRCA2 have been found to be significantly hypermethylated in Lichen Sclerosis (LS) and Squamous Cell Carcinoma (SCC). And RASSF1A has displayed an interesting methylation pattern in the post transcription region, where the frequency of methylation significantly decreased from normal tissue to LS tissue, but then dramatically increased to SCC. H19 failed to show any changes in the methylation pattern with methods tested. This was most likely due to interference of primer dimers by SYBR-green in the real-time PCR analyses. Instead of using the same promoter sequence reported by previous papers, extended estimated promoter regions and partial transcription regions were obtained from Genome Browser and additional CpG island sites have been studied in current project. Methylation patterns have been detected that differ from the literature in these genes. These results may provide more information to find a more precise active promoter region and epigenetic involved sequences for future research.

LINE-1s (Long interspersed elements or L1s) and Alus are highly successful non-long terminal repeat retrotransposons with copy numbers of ~520,000 and >1 million within the human genome, respectively. They are associated with human genetic variation and genomic rearrangement. Although they are abundant throughout primate genomes, their propagation strategy remains poorly understood. The recently released human and chimpanzee draft genome sequences provide the opportunity to compare the human genome with the chimpanzee genome. Thus, we were able to assess how these elements expanded in primate genomes and how they create genomic instability during their integration into the host genome as well as subsequent post-insertion recombination between elements. To understand the expansion of Alu elements, we first analyzed the evolutionary history of the AluYb lineage which is one of most active Alu lineages in the human genome. We suggest that the evolutionary success of Alu elements is driven at least in part by stealth driver elements that maintain low retrotransposition activity over extended periods of time and occasionally produce short-lived hyperactive copies responsible for the formation and remarkable expansion of Alu elements within the genome. Second, we conducted a detailed characterization of chimpanzee-specific L1 subfamily diversity. Our results showed that L1 elements have experienced different evolutionary fates in humans and chimpanzees lineages. These differential evolutionary paths may be the result of random variation or the product of competition between L1 subfamily lineages. Third, we report 50 deletion events in human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of human genomic sequence and ~15 kb of chimpanzee genomic sequence. This study provides the basis for developing models of the mechanisms for small and large L1 insertion-mediated deletions. Fourth, we analyzed the magnitude of Alu recombination-mediated deletions in the human lineage subsequent to the human-chimpanzee divergence. We identified 492 human-specific deletions (for a total of ~400 kb of sequence) attributable to this process. The majority of the deletions coincide with known or predicted genes, which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees.