Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.

<p>In the post-genomics era, the sheer volume of data is overwhelming without appropriate tools for data integration and analysis. Studying genomic sequences in the context of other related genomic sequences, i.e. comparative genomics, is a powerful technique enabling the identification of functionally interesting sequence regions based on the principal that similar sequences tend to be either homologous or provide similar functionality.</p> <p>Costs associated with full genome sequencing make it infeasible to sequence every genome of interest. Consequently, simple, smaller genomes are used as model organisms for more complex organisms, for instance, Mouse/Human. An annotated model organism provides a source of annotation for transcribed sequences and other gene regions of the more complex organism based on sequence homology. For example, the gene annotations from the model organism aid interpretation of expression studies in more complex organisms.</p> <p>To assist with comparative genomics research in the Arabidopsis/Brassica (Thale-cress/Canola) model-crop pair, a web-based, graphical genome browser (BioViz) was developed to display short Brassica genomic sequences in the context of the Arabidopsis model organism genome. This involved the development of graphical representations to integrate data from multiple sources and tools, and a novel user interface to provide the user with a more interactive web-based browsing experience. While BioViz was developed for the Arabidopsis/Brassica comparative genomics context, it could be applied to comparative browsing relative to other reference genomes.</p> <p>BioViz proved to be an valuable research support tool for Brassica / Arabidopsis comparative genomics. It provided convenient access to the underlying Arabidopsis annotation, allowed the user to view specific EST sequences in the context of the Arabidopsis genome and other related EST sequences. In addition, the limits to which the project pushed the SVG specification proved influential in the SVG community. The work done for BioViz inspired the definition of an opensource project to define standards for SVG based web applications and a standard framework for SVG based widget sets.</p>

Recent advances in DNA sequencing technology have enabled entire genomes to be sequenced quickly and accurately, resulting in an exponential increase in the number of organisms whose genome sequences have been elucidated. While the genome sequence of a given organism represents an important starting point in understanding its physiology, the functions of the protein products of many genes are still unknown; as such, computational methods for studying protein function are becoming increasingly important. In addition, this wealth of genomic information has created an unprecedented opportunity to compare the protein content of different organisms; among other applications, this can enable us to improve taxonomic classifications, to develop more accurate diagnostic tests for identifying particular bacteria, and to better understand protein content relationships in both closely-related and distantly-related organisms.<p> This thesis describes the design, evaluation, and use of a program called Proteome Subtraction and Intersection (PSI) that uses an idea called genome subtraction for discovering protein-phenotype relationships and for characterizing differences in protein content in groups of organisms. PSI takes as input a set of proteomes, as well as a partitioning of that set into a subset of "included" proteomes and a subset of "excluded" proteomes. Using reciprocal BLAST hits, PSI finds orthologous relationships among all the proteins in the proteomes from the original set, and then finds groups of orthologous proteins containing at least one orthologue from each of the proteomes in the "included" subset, and none from any of the proteomes in the "excluded" subset.<p> PSI is first applied to finding protein-phenotype relationships. By identifying proteins that are present in all sequenced isolates of the genus <i>Lactobacillus</i>, but not in the related bacterium <i>Pediococcus pentosaceus</i>, proteins are discovered that are likely to be responsible for the difference in cell shape between the lactobacilli and <i>P. pentosaceus</i>. In addition, proteins are identified that may be responsible for resistance to the antibiotic gatifloxacin in some lactic acid bacteria.<p> This thesis also explores the use of PSI for comparing protein content in groups of organisms. Based on the idea of genome subtraction, a novel metric is proposed for comparing the difference in protein content between two organisms. This metric is then used to create a phylogenetic tree for a large set of bacteria, which to the author's knowledge represents the largest phylogenetic tree created to date using protein content. In addition, PSI is used to find the proteomic cohesiveness of isolates of several bacterial species in order to support or refute their current taxonomic classifications.<p> Overall, PSI is a versatile tool with many interesting applications, and should become more and more valuable as additional genomic information becomes available.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

The rapid development of large-scale genomic sequencing has dramatically changed the field of genetics, in part through the development of comparative genomics. Fungal comparative genomics is particularly powerful given the large number of genomes currently available, their compact architecture, and their relative ease of genetic manipulation. Fungal comparative genomics was employed in this work to address two related questions. First, it was used along with computational thermodynamic methods to predict non-coding RNA (ncRNA) in Saccharomyces cerevisiae. Sets of positive and negative control genes were evaluated to determine the effect of window sizes and step sizes on the sensitivity of ncRNA identification. The approach was then applied to predict ncRNA genes on chromosome 6 of S. cerevisiae and S. bayanus. Northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data were used to test the predictions. Strong experimental evidence was accumulated for four new ncRNA genes. Potential structural elements in the 5' and 3' untranslated regions (UTRs) of six annotated protein-coding genes were also identified. This work shows that thermodynamic approaches, coupled with comparative genomics, are powerful tools for predicting structural ncRNA. Second, comparative genomic approaches were employed to identify a non-reciprocal transfer event from Cryptococcus neoformans var. grubii to var. neoformans ~2 million years ago involving a 14 gene (~40 kb) region. The majority of clinical and environmental var. neoformans strains from around the world contain this sequence obtained from var. grubii. The introgression event likely occurred via an incomplete inter-varietal sexual cycle creating a hybrid intermediate where mobile elements common to both lineages mediated the exchange. The subsequent duplication in laboratory strains of a fragment of this same genomic region supports evolutionary theories that instabilities in subtelomeric regions promote adaptive evolution through gene amplification and subsequent adaptation. These data indicate that DNA exchange between closely related sympatric varieties or species may be a recurrent theme in the evolution of fungal species. It further suggests that while evolutionary divergence is the primary force driving speciation, rare introgression events also play a potentially important role. / Dissertation

<p>Allelic imbalance, in which two alleles at a given locus exhibit differences in gene expression, chromatin composition and/or protein binding, is a widespread phenomenon in the human and other complex genomes. Most examples concern individual loci located more or less randomly around the genome and thus imply local and gene-specific mechanisms. However, genomic or chromosomal basis for allelic imbalance is supported by multi-locus examples such as those exemplified by domains of imprinted genes, spanning ~1-2 Mb, or by X chromosome inactivation, involving much of an entire chromosome. Recent studies have shown that genes on the two female X chromosomes exhibit a breadth of expression patterns ranging from complete silencing of one allele to fully balanced biallelic expression. Although evidence for heritability of allele-specific chromatin and expression patterns exists at individual loci, it is unknown whether heritability is also reflected in the chromosome-wide patterns of X inactivation.</p><p>The aim of this thesis is to elucidate the extent to which the widespread variable patterns of allelic imbalance on the human X chromosome in females are under genetic control and how access of the transcription machinery to the human inactive X chromosome in females is determined at a genomic level. For the set of variable genes examined in this study, the absence or presence of expression appears to be stochastic with respect to the population rather than abiding by strict genetic rules. Furthermore, variable gene expression that I have detected even among multiple clonal cell lines derived from a single individual suggests fluctuation in transcriptional machinery engagement. I find that, although expression at most genes on the human inactive X chromosome is repressed as a result of X inactivation, a number of loci are accessible to the transcriptional machinery. It appears that RNA Polymerase II is present at alleles on the inactive X even at the promoters of several silenced genes, indicating a potential for expression. </p><p>This thesis embodies a transition in the field of human X chromosome inactivation from gene by gene approaches used in the past to utilizing high-throughput technologies and applying follow-up analytic techniques to draw upon the vast data publicly available from large consortia projects.</p> / Dissertation

MicroRNAs are a large family of 21-26 nucleotide non-coding RNAs with a role in the post-transcriptional regulation of gene expression. In recent years, microRNAs have been proposed to play a significant role in the expansion of organism complexity. MicroRNAs are expressed in a cell or tissue-specific manner during embryonic development, suggesting a role in cellular differentiation. For example, Let-7 is a metazoan microRNA that acts as developmental timer between larval stages in C. elegans. We conducted a comparative study that determined the distribution of microRNA families among metazoans, including the identification of new family members for several species. MicroRNA families appear to have evolved in bursts of evolution that correlate with the advent of major metazoan groups such as vertebrates, eutherians, primates and hominids. Most microRNA families identified in these organisms appeared with or after the advent of vertebrates. Only a few of them appear to be shared between vertebrates and invertebrates. The distribution of these microRNA families supports the idea that at least one whole genome duplication event (WGS) predates the advent of vertebrates. Gene ontology analyses of the genes these microRNA families regulate show enrichments for functions related to cell differentiation and morphogenesis. MicroRNA genes appear to be under great selective constraints. Identification of conserved regions by comparative genomics allows for the computational identification of microRNAs. We have identified and characterized ultraconserved regions between the genomes of the honey bee (Apis mellifera) and the parasitic wasp (Nasonia vitripennis), and developed a strategy for the identification of microRNAs based on regions of ultraconservation. Ultraconserved regions preferentially localize within introns and intergenic regions, and are enriched in functions related to neural development. Introns harboring ultraconserved elements appear to be under negative selection and under a level of constraint that is higher than in their exonic counterparts. This level of constraint suggests functional roles yet to be discovered and suggests that introns are major players in the regulation of biological processes. Our computational strategy was able to identify new microRNA genes shared between honey bee and wasp. We recovered 41 of 45 previously validated microRNAs for these organisms, and we identified several new ones. A significant fraction of these microRNA candidates are located in introns and intergenic regions and are organized in genomic clusters. Expression of 13 of these new candidates was verified by 454 sequencing.

Although the completion of the Human Genome Project helps develop efficient treatment/prevention programs, it will raise new and non-trivial public health issues. Many of these issues fall under the professional purview of health educators. Yet, no studies have evaluated if health educators (HEs) are ready to adopt genomic competencies into health promotion. This dissertation addresses this issue by examining three research questions in three separate studies: 1) Why must HEs develop genomic competencies? 2) What are HEs’ knowledge of, and attitudes toward genomic competencies? And 3) what is HEs’ likelihood of adopting genomic competencies into health promotion? The first theoretical study proposed five arguments supporting the need for HEs to develop their genomic competencies and integrate public health genomics into health promotion. These arguments touched on various dimensions of HEs’ professional goals and ranged from professional responsibilities and competencies, to the availability of funding for genomic-related research or interventions and opportunities for future employment. For the second study, a web-based survey was developed and distributed to all members of four major health education organizations. A total of 1,925 HEs’ completed the survey and 1,607 responses were utilized in the final analysis. This study indicated that participants had deficient knowledge and unfavorable attitudes toward the CDCproposed genomic competencies. In the third study, a theoretical model was developed to predict HEs’ likelihood to incorporate genomic competencies into their practice. Using techniques from Structural Equation Modeling (SEM), the model was tested with the same data of the second study. Findings supported the proposed theoretical model. While genomic knowledge, attitudes, and self-efficacy were significantly associated with HEs’ likelihood to incorporate genomic competencies into their practice, attitudes was the strongest predictor of likelihood. In summary, these studies indicated that participating HEs had deficient genomic knowledge, unfavorable attitudes toward a set of CDC-proposed genomic competencies, and low likelihood to adopt genomic competencies into health promotion. Relevant training should be developed and advocated. As the SEM analysis results indicated the survey findings supported the proposed theoretical model, which can be utilized to steer future training for HEs.

The major histocompatibility complex (MHC) is a genomic region containing genes of immunomodulatory importance. MHC class I genes encode cell-surface glycoproteins that present peptides to circulating T cells, playing a key role in recognition of self and non-self. Studies of MHC loci in vertebrates have examined levels of polymorphism and molecular evolutionary processes generating diversity. The bovine MHC (BoLA) has been associated with disease susceptibility, resistance and progression. To delineate mechanisms by which MHC class I genes evolved to function optimally in a species like cattle, it is necessary to study genomic organization of BoLA to define gene content, and investigate characteristics of expressed class I molecules. This study describes development of a physical map of BoLA class I region derived from screening two BAC libraries, isolating positive clones and confirming gene content, order and chromosomal location through PCR, novel BAC end sequencing techniques, and selected BAC shotgun cloning and/or sequencing and FISH analysis. To date, this is the most complete ordered BAC array encompassing the BoLA class I region from the class III boundary to the extended class I region. Characterization of a frameshift allele exhibiting trans-species polymorphism in Bos and Bison by flow cytometry, real-time RT-PCR, 1D and 2D gel analysis is also described. This frameshift allele encodes an early termination signal within the antigen recognition site (ARS) of exon 3 of the BoLA BSA-Adel class I gene predicting a truncated class I protein that is soluble. An ability to assess MHC diversity in populations and provision of animals with defined MHC haplotypes and genetic content for experimental research is necessary in developing a basis upon which to build functional studies to elucidate associations between haplotype and disease in bovids. The BoLA class I region is immunologically important for disease association studies in an economically important species. This study provides knowledge of gene content and organization within the class I MHC region in cattle, providing a template for more detailed analysis and elucidation of complex disease associations through functional genomics and comparative analysis, as well as evolution of the MHC in bovids to optimize a population’s immune response.

Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this study, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to identify systematically chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. The TLR family represents evolutionarily conserved components of the patternrecognizing receptors (PRRs) of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs). TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs play important roles in the activation of pro-inflammatory cytokines, chemokines and modulation of antigen-specific adaptive immune responses. To date, nine TLRs have been reported in chicken, along with a non-functional TLR8. Two non-mammalian TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The objectives of this study were to determine if there is the existence of chicken genes homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After searching the chicken genome sequence and EST database, a novel chicken TLR homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of potential PAMP binding sites within LRR insertions showed that CpG DNA is the putative ligand of this receptor.