Through their regulation of gene expression and cell growth, auxins affect many aspects of plant growth and development. One mechanism through which auxin homeostasis is achieved is by conjugating indole-3-acetic acid (IAA, the most common natural form of auxin) to amino acids or sugars. The Arabidopsis mutant ilr2-1 was isolated as an I&barbelow;AA-L&barbelow;eucine r&barbelow;esistant mutant that retains wild-type sensitivity to free IAA. ilr2-1 is resistant to IAA-Leu and IAA-Phe, but not to other conjugated forms of IAA. It is also resistant to cobalt and manganese. ilr2-1 has fewer lateral roots than wild type, which can be rescued by exogenous IAA conjugates; and it has a shorter root than wild type, which can be rescued by exogenous metals, including cobalt. Using a map-based approach, I cloned the ILR2 gene, which encodes a novel protein that is polymorphic between Arabidopsis ecotypes. A T-DNA insertion in the second form of ILR2, ilr2-2, results in cobalt supersensitivity. The ILR2 transcript appears to be expressed at very low levels, and accumulates in aerial tissues and in response to exogenous cobalt. Auxin resistant mutant screens have provided valuable information about how auxin is sensed and regulated; they also suggest that a considerable part of auxin homeostasis is dependent on negative regulation. To identify these negative regulators, we developed a genetic screen that allows the identification of three a&barbelow;ux&barbelow;in s&barbelow;upersensitive (axs) mutants. These mutants exhibit abnormal responses in conditions leading to auxin accumulation, such as auxin transport inhibitors and growth at high temperatures. We used recombination mapping to identify the genes defective in these mutants. The axs1 mutant that is resistant to low concentrations of auxin transport inhibitors, and the axs2 mutant that has a higher number of lateral roots than wild type. Both mutations were mapped, based on a defective root-curling phenotype, to the region in chromosome 1 contained between the markers nga280 and nga111; this mapping location may represent a QTL involved in root-curling. The axs3 mutant, supersensitive to high temperature, was mapped to the region in chromosome I contained between the markers nga62 and nga280. The analysis of mutants such as axs3 is expected to contribute to our understanding of how auxin levels are spatially and temporally regulated in the plant.

DNA sequences sampled from a genetic locus within a population are related by a genealogy. If there is no recombination within the locus, each pair of sequences is descended from some ancestral sequence, one of which is the most recent common ancestor of the entire sample. Past demography shapes this genealogy since the branch lengths depend on the size history of the population. For this reason, observed distributions of allelic types carry information about the population's demographic history. Because of their abundance and relative ease of typing, microsatellites, or short tandem repeats, represent a useful class of loci for the study of demography. This thesis investigates the properties of the imbalance index beta, a microsatellite-based statistic constructed for demographic inference. Simulated data sets are used to explore the sampling properties of beta and to compare its performance to that of other statistics available in the literature. Tests based on these statistics are applied to samples of microsatellite loci from human populations, and the results are interpreted in light of recent hypotheses concerning the evolution of modern humans.

Terpenes are one of the largest groups of natural products. Sesquiterpenes (15-carbon terpenes) are secondary metabolites produced from farnesyl pyrophosphate (FPP). Although these compounds are normally found in plants, production levels are too low for commercial use. As a result, we have begun engineering the metabolic pathways of Saccharomyces cerevisiae to accumulate sesquiterpenes. Epi-cedrol is a sesquiterpene identified from Artemisia annua, which was used as a reporter molecule to monitor sesquiterpene production in our engineered yeast strains. Through manipulations of the ergosterol pathway, we were able to increase sesquiterpene production from 0.090 mg/L (wild type yeast) to 1.000 mg/L in yeast strains that overexpress the truncated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme (HMG1) gene in an erg9 background. This yeast strain also increased the in vivo production of farnesol and trans-nerolidol, the hydrolyzed products of FPP. Likewise, the use of a different resin to isolate the epi-cedrol allowed a further increase to 1.883 mg/L in this yeast strain. Part II describes a valuable alternative to the specifically designed yeast strains described in Part I of this thesis by producing the yellow compound, 4,4'-diaponeurosporene. 4,4'-Diaponeurosporene is a deep yellow triterpene carotenoid identified in Staphylococcus aureus. CrtM and CrtN are two genes that are cotranscribed to convert FPP to this pigment. Because the CrtM/CrtN pair converts FPP to a colored compound, the color of yeast cells expressing these genes will provide a visual indication of the amount of FPP that can be diverted to foreign product biosynthesis. Finally, Part III of this thesis describes the use of our engineered yeast strains to characterize Medicago truncatula sesquiterpene synthase products. Now that significant amounts of genomic and cDNA sequences have been determined for many organisms, genes of interest can be identified by comparison to other genes in that family. Seven genes were obtained and cloned into yeast expression vectors, transformed into our yeast strains and the products isolated. Several compounds were identified using GC-MS analysis.

In bacteria and viruses, recombination and horizontal gene transfer (HGT) permit the direct exchange or acquisition of DNA into the genome from species other than the "parent". Two major challenges face attempts at reconstructing non-treelike evolution: (1) multiple sources of gene tree incongruence exist, making various reconciliation scenarios possible, and (2) reconstructing a given reconciliation scenario is computationally hard. In this thesis, we address the latter problem, describing new methods for reconstructing recombination and HGT events: RECOMP and RIATA-HGT. RECOMP is a recombination detection method, faster than all existing algorithms and as accurate as the best. RIATA-HGT is the first polynomial-time heuristic that reconstructs unrestricted HGT events based on gene tree incongruence. We also describe two studies characterizing the sources of statistical error in phylogenetic incongruence: ortholog inference and phylogeny estimation. These studies identify computational problems whose revision will improve the overall accuracy of phylogenetic incongruence-based, non-treelike evolution reconstruction tools.

This dissertation introduces a novel approach for addressing the complexities of mapping a complex disease by adjusting a Bayesian Model Selection method. Mapping the genes for a complex disease, such as Rheumatoid Arthritis, involves finding multiple genetic loci that may contribute to the onset of the disease. Pairwise testing of the loci leads to the problem of multiple testing. To avoid multiple tests, one can look at haplotypes, or linear sets of loci, but this results in a contingency table with sparse counts, especially when using marker loci with multiple alleles. In order to jointly consider all loci in the problem, we applied a Hierarchical Bayesian Model Selection method to a conditional logistic regression model used in gene mapping. We chose Stochastic Search Variable Selection for its use of latent indicator variables to indicate those covariates, in this case genes, important to the model. We extended the latent variable structure to mirror genetics through a latent allele indicator conditional on a latent locus indicator. We also examined using a prior correlation structure on the allele coefficients that mirrors linkage disequilibrium, a between-locus genetic correlation structure. Ultimately, we ruled out the usefulness of a dependent covariance structure on the prior for allele main effects, but we developed a preliminary method of fitting a positive definite matrix to data based on adjusting the kriging covariance functions commonly used in geostatistics or spatial statistics. We developed a Metropolis-within-Gibbs algorithm to sample our gene suggestion posterior, and evaluated the algorithm's performance on simulated data and completed the research with application to real data, searching for genes associated with Rheumatoid Arthritis. On simulated data, we found that our method successfully recognized disease loci and nondisease loci. Despite complications when analyzing the real data, our method did indicate the genes more strongly associated with Rheumatoid Arthritis.

I examined sequences of 3 microsatellite loci that were derived from Polistes bellicosus investigated their evolution in 78 species from four tribes in the Polistinae subfamily of wasps. I found that the basic repeat structure of the microsatellite repeat region at these three loci was conserved across species at the subgenus level. Besides changes in the number of repeats, base substitutions were the dominant mutations in the repeat regions. The tribe from which the loci were isolated had longer repeat regions and more perfect repeats that the tribes at the same loci. At microsatellite flanking regions, I found that a high proportion of insertions duplicated 1-4 bases of their preceding sequences. Microsatellite repeats may originate from these such little insertions.

By studying the rate of recombination between genetic markers and disease genes with linkage analysis, scientists have successfully mapped the locations of disease-influencing genes to within one centiMorgan. However, one centiMorgan corresponds to a sequence of about one million base pairs of DNA, which is prohibitively large for a physical search for a specific gene. Therefore, other genetic mapping techniques are needed to define search regions that are small enough for physical mapping techniques to be feasible. One such method is called linkage disequilibrium mapping. Linkage disequilibrium can serve as a complement, or even an alternative, to linkage analysis. It is capable of estimating genetic distances that are as small as tens of kilobases of DNA, a great improvement over the resolution of linkage analysis. However, one must describe the joint transmission of disease genes and linked marker loci through many generations in order to use linkage disequilibrium for genetic mapping purposes. This thesis examines two classes of population models, Galton-Watson branching processes and Moran/Coalescent models, within the framework of linkage disequilibrium. That is, it uses moments of allele frequencies derived from these models to form approximate likelihood functions for the recombination rate. These likelihoods make it possible to estimate the location of a disease-influencing mutation, particularly when the likelihoods from several markers within a small region of DNA are combined to form a composite likelihood. Application of this composite likelihood methodology to both simulated and published data demonstrates that linkage disequilibrium mapping can be successfully used for fine-scale mapping purposes.

Current population genetics literature presumes a lack of population structure in microorganisms due to their large population sizes, ubiquitous dispersal, and absence of geographic barriers. Contrary to this, we found evidence of great population subdivision in the social amoeba, Dictyostelium discoideum. Pairwise values of Fst and Rst were significant between samples collected from sites separated by distances from 100m--10,000km (P < 0.05 after sequential Bonferroni). Three- and two-level analyses of molecular variance (AMOVA) confirmed the existence of population structure (Fst 0.30, Rst = 0.48, P < 0.001 for three-level; Fst = 0.297, Rst = 0.474, P < 0.001 for two-level). Further, linearized pairwise Fst values were significantly correlated with natural logarithm of geographic distance (r = 0.471, P < 0.012) indicating isolation by distance. Genotypic diversity of populations remained moderate (Nei (1987) average gene diversity ∼0.50) in spite of the decreased population diversity expected with such Fst and Rst values.

The Drosophila inebriated (ine) gene encodes a putative transporter (Ine) that shares high homology to members of the Na+/Cl- dependent neurotransmitter transporter family. Mutations in the ine gene were found to cause increased neuronal excitability. Research documented in this thesis demonstrated that ine also confers defective osmotic stress response, confirming the dual roles played by certain members of this family in regulating both neuronal excitability and osmotic stress response. In addition, from further investigation of the neuronal phenotypes of ine mutants it was discovered that Ine might act in short-term to affect neuronal excitability, that the transporter can exert its function from either neurons or glia, and that the two isoforms of the transporter, Ine-P1 and Ine-P2, which are identical in major portion of their sequence but differ in their N termini, are both capable of their function in the absence of the other, although the former functions more efficiently. Furthermore, ine overexpression causes phenotypes that closely resemble those of mutants with defective sodium channels. These phenotypes include delayed onset of long-term facilitation, suppression of the leg-shaking phenotypes of Shaker , temperature sensitive paralysis, enhancement of the paralytic (para) mutation, increased failure rate of transmitter release at the larval neuromuscular junction, reduced amplitude of larval nerve compound action potential and failure of compound action potential at restrictive temperature. Taken together, these observations raise the possibility that ine might be involved in a signaling pathway that regulates neuronal sodium channels.

NF-kappaB transcription factors are central to the regulation of many vital processes including immune response. It is known from microarray measurements and clustering methods that NF-kappaB dependent genes in humans are expressed in functionally distinct "waves". This research helps determine how the expression of these sequences is a consequence of their structure, molecular constitution and evolution. This thesis identifies location of TF-binding sites and consensus regions in the DNA sequences that are upregulated by NF-kappaB and examines their structure. This project uses a variety of tools and databases available for sequence analysis including Blast, BLAT, MATCH, GeneBee, BioProspector and MEME. This analysis is one aspect of the larger 'Investigation of NF-kappaB Pathways' project underway at UTMB, Galveston and Rice University. Future promoter analysis of these results will verify the location of functional regulatory sites, thereby enabling us to postulate and verify a model governing expression of NF-kappaB dependent genes.