In the past decade, automatisation has led to an immense increase of data in biology. Next generation sequencing techniques will produce a vast amount of sequences across all species in the coming years. In many cases, identifying the function and biological role of a protein from its sequence can be a complicated and time-intensive task. The identification of a protein's interaction partners is a tremendous help for understanding the biological context in which it is involved. In order to fully characterise a protein-protein interaction (PPIs), it is necessary to know the three-dimensional structure of the interacting partners. Despite optimisation efforts from projects such as the Protein Structure Initivative, determining the structure of a protein through crystallography remains a time- and cost-intensive procedure. The primary aim of the research described in this dissertation was to produce a World Wide Web resource that facilitates visual exploration and validation (or questioning) of data derived from functional genomics experiments, by building upon existing structural information about direct physical PPIs. Secondary aims were (i) to demonstrate the utility of the new resource, and (ii) its application in biological research. We created a database that emphasises specifically the intersection between the PPIs-results emerging from the structural biology and functional genomics communities. The BISC database holds BInary SubComplexes and Modellable Interactions in current functional genomics databases (BICS-MI). It is publicly available at hyyp://bisc.cse.ucsc.edu. BISC is divided in three sections that deliver three types of information of interest to users seeking to investigate or browse PPIs. The template section (BISCHom and BISCHet) is devoted to those PPIs that are characterised in structural detail, i.e. binary SCs extracted from experimentally determined three-dimensional structures. BISCHom and BISCHet contain the homodimeric (13,583 records) and heterodimeric (5612 records) portions of these, respectively. Besides interactive, embedded Jmol displays emphasising the interface, standard information and links are provided, e.g. sequence information and SPOP classification for both partners, interface size and energy scores (PISA). An automated launch of the MolSurfer program enables the user to investigate electrostatic and hydrophobic correlation between the partners, at the inter-molecular interface. The modellable interactions section (BISC0MI) identifies potentially modellable interactions in three major functional genomics interaction databases (BioGRID), IntAct, HPRD). To create BISC-MI all PPIs that are amenable to automated homology modelling based on conservative similarity cut-offs and whose partner protein sequences have recrods in the UniProt database, have been extracted. The modellable interaction services (BISC-MI Services) section offers, upon user request, modelled SC-structures for any PPIs in BISC-MI. This is enabled through an untomated template-based (homology) modelling protocol using the popular MODELLER program. First, a multiple sequence alignment (MSA) is generated using MUSCLE, between the target and homologous proteins collected from UniProt (only reviewed proteins from organisms whose genome has been completely sequenced are included to find putative orthologs). Then a sequence-to-profile alignment is generated to integrate the template structure in the MSA. All models are produced upon user request to ensure that the most recent sequence data for the MSAs are used. Models generated through this protocol are expected to be more accurate generally than models offered by other automated resources that rely on pairwise alignments, e.g. ModBase. Two small studies were carried out to demonstrate the usability and utility of BISC in biological research. (1) Interaction data in functional genomics databases often suffers from insufficient experimental and reporting standards. For example, multiple protein complexes are typically recorded as an inferred set of binary interactions. Using the 20S core particle of the yeast proteasome as an example, we demonstrate how the BISC Web resource can be used as a starting point for further investigation of such inferred interactions. (2) Malaria, a mosquito-borne disease, affects 3500-500 million people worldwide. Still very little is known about the malarial parasites' genes and their protein functions. For Plasmodium falciparum, the most lethal among the malaria parasites, only one experimentally derived medium scale PPIs set is available. The validity of this set has been doubted in the the malarial biologist community. We modelled and investigated eleven binary interactions from this set using the BISC modelling pipeline. Alongside we compared the BISC models of the individual partners to those obtained from ModBase.

Microsatellites are short (1-6bp long) highly polymorphic tandem repeats, found in all genomes analyzed so far. Popular genetic markers for many applications including population genetics, pedigree analysis, genetic mapping and linkage analysis, some microsatellites also can cause a variety of human neurodegenerative diseases and may act as agents of adaptive evolution through the regulation of gene expression. As a consequence of these diverse uses and functions, the mutational and evolutionary dynamics of microsatellite sequences have gained much attention in recent years. Mostly, the focus of studies investigating microsatellite evolution has been to develop more refined evolutionary models for estimating parameters such as genetic distance or linkage disequilibrium. However, there is an incentive in using our understanding of the evolutionary processes that affect these sequences to examine the functional implications of microsatellite evolution. What has emerged from nearly two decades of study are highly complex mutational dynamics, with mutation rates varying across species, loci and alleles, and a multitude of potential influences on these rates, most of which are not yet fully understood. The increasing availability of whole genome sequences has immensely extended the scope for studying microsatellite evolution. For example, where once it was common to examine single loci, it is now possible to examine microsatellites using genome wide approaches. In the first part of my dissertation I discuss approaches and issues associated with detecting microsatellites in genomic data. In Chapter 2 I undertook a meta-analysis of studies investigating the distribution of microsatellites in yeast and showed that studies comparing the distribution of microsatellites in genomic data can be fraught due to the application of different definitions for microsatellites by different investigators. In particular, I found that variation in how investigators choose the repeat unit size of a microsatellite, handle imperfections in the array and especially the choice of minimum array length used, leads to a large divergence in results and can distort the conclusions drawn from such studies, particularly where inter-specific comparisons are being made. In a review of the currently available suite of bioinformatics tools (Chapter 3), I further showed that this bias extends beyond a solely theoretical controversy into a methodological issue because most software tools not only incorporate different definitions for the key parameters used to define microsatellites, but also employ different strategies to search and filter for microsatellites in genomic data. In this chapter I provide an overview of the available tools and a practical guide to help other researchers choose the appropriate tool for their research purpose. In the second part of my thesis, I use the analytical framework developed from the previous chapters to explore the biological significance of microsatellites exploiting the well annotated genome of the model organism Saccharomyces cerevisiae (baker’s yeast). Several studies in different organisms have indicated spatial associations between microsatellites and individual genomic features, such as transposable elements, recombinational hotspots, GC-content or local substitution rate. In Chapter 4, I summarized these studies and tested some of the underlying hypotheses on microsatellite distribution in the yeast genome using Generalized Linear Models (GLM) and wavelet transformation. I found that microsatellite type and distribution within the genome is strongly governed by local sequence composition and negative selection in coding regions, and that microsatellite frequency is inversely correlated with SNP density reflecting the stabilizing effect point mutations have on microsatellites. Microsatellites may also be markers for recent genome modifications, due to their depletion in regions nearby LTR transposons, and elements of potential structural importance, since I found associations with features such as meiotic double strand breaks, regulatory sites and nucleosomes. Microsatellites are subject to local genomic influences, particularly on small (1-2kb) scales. Although, these local scale influences might not be as dominant as other factors on a genome-wide scale they are certainly of importance with respect to individual loci. Analysis of locus conservation across 40 related yeast strains (Chapter 5) showed no bias in the type of microsatellites conserved, only a negative influence of coding sequences, which supports again the idea that microsatellites evolve neutrally. Polymorphism was rare, and despite a positive correlation with array length, there was no relationship with either genomic fraction or repeat size. However, the analysis also revealed a non-random distribution of microsatellites in genes of functionally distinct groups. For example, conserved microsatellites (similar to general microsatellites in yeast) are mostly found in genes associated with the regulation of biological and cellular processes. Polymorphic loci show further an association with the organization and biogenesis of cellular components, morphogenesis, development of anatomical structures and pheromone response, which, is absent for monomorphic loci. Whether this distribution is an indication of functionality or simply neutral mutation (e.g. genetic hitch-hiking) is debatable since most conserved microsatellites, particularly variable loci, are located within genes that show low selective constraints. Overall, microsatellites appear as neutrally evolving sequences, but owing to the sheer number of loci within a single genome, individual loci may well acquire some functionality. More work is definitely needed in this area, particularly experimental studies, such as reporter-gene expression assays, to confirm phenotypic effects.

Atopic asthma is a complex disease process that has a significant social, personal and economic burden across all ages. Leukotriene-receptors are involved in the cascade of inflammation that may result in symptoms of atopy and asthma. Two leukotriene receptors have been identified in the lung. The cysteinyl leukotriene receptor 1 and cysteinyl leukotriene receptor 2 genes (i.e., CYSLTR1 and CYSLTR2) have been sequenced, and a number of single nucleotide polymorphisms (SNPs) within these genes have been identified.The purpose of this study was to: (1) Determine the relationship between CYSLTR1 genotypes, CYSLTR2 genotype, atopy, elevated IgE level, and eosinophilia, (2) Determine the relationship between CYSLTR1 genotypes, CYSLTR2 genotype, asthma, and atopic asthma, and (3) Determine the degree of interaction between CYSLTR2 genetic variation and gender in atopic asthma.Nested within two sub-studies of the Tucson Epidemiological Study of Airway Obstructive Disease (TESAOD) study, a prospective longitudinal cohort, 853 individuals were entered into this study. Study criteria included Non-Hispanic white adults, who consented to genetic testing in the two sub-studies. Tagging SNPs of the CYSLTR1 and CYSLTR2 genes were genotyped. Serum IgE status and eosinophilia were obtained from existing dataset. Questionnaires collected in the parent study were used to obtain demographic and clinical data.SNP rs321006 in the CYSLTR1 gene was associated with atopy among Non-Hispanic white women. Assuming a recessive model, among female Non-Hispanic white adults, the odds of having rs321073 CC genotype was 5.82 times higher among those with atopic asthma than those without atopic asthma. No gene by gender interaction was found between SNP of interest in CYSLTR2 and atopic asthma. Genetic association of SNPs rs321006 with atopy and rs321073 with atopic asthma are novel findings to date.Implications for nurses, clinicians, and scientists include better understanding of associations of these genetic variations with asthma, atopy, and atopic asthma that can generate further inquiry into other mechanisms of atopic asthma. These novel genetic associations with atopy and atopic asthma may have the potential for personalized medicine that might afford patients with appropriate treatment based on their genotype.

Rice is one of the most important crops in the world and is the first whose genome was completely sequenced. This landmark accomplishment placed O. sativa as a leading model in plant biology, especially for cereals. The genus Oryza includes 23 species, two of them independently domesticated in Asia and Africa. Wild species of Oryza contain a reservoir of useful agronomical traits which could be exploited for the benefit of rice agriculture, which is facing global problems as other crops, mainly due to a rampant increase in the human population and progressive deterioration of soils and water supplies. The Oryza Mapping Alignment Project has opened great opportunities to tap the genetic potential encapsulated in these species. Four BAC libraries generated from the African species of Oryza: O. barthii, O. glaberrima (AA genome), O. punctata (BB genome) and O. brachyantha (FF genome) were fully characterized and shown to provide enough coverage to represent their respective genomes. BAC clones from these libraries were fingerprinted and end-sequenced to assemble physical maps that were heavily manually edited using the sequence of O. sativa as a reference genome. The physical maps showed high coverage for all the species across all chromosomes. Both, BAC libraries and physical maps were used to investigate synteny and structural variation. The four species show high colinearity to the reference genome, although synteny perturbations were detected, including contractions, expansions, and putative inversions and translocations, which potential have an important impact in the evolution of these species.

A role for gene variants in regulating the pharmacokinetics of systemically available toxicants has not yet been established. A panel of 18 genetically-diverse inbred mouse strains was used to determine the range of total exposure kinetic parameters in blood and bone marrow following a single oral administration of benzene (100 μg/kg) to male and female mice. Large ranges in several pharmacokinetic parameters were found when data from blood and bone marrow were analyzed. AUC and CL_F pharmacokinetic parameters in blood and bone marrow pharmacokinetics were strikingly different as were these parameters in males and females. Final clearance (CL_F) was found to be the most statistically robust pharmacokinetic parameter as it accounted for exposure of the matrix (AUC) and normalized for dose variations among the strains. The CL_F values in blood and bone marrow used for haplotype association mapping showed 331 and 164 quantitative trait loci with statistical significance, respectively (male mice; -logP>4). Two loci were found to be shared between males and females QTL bone marrow data sets and one common locus was found for male blood and bone marrow data. No overlap was found among blood QTL in males and females (or between blood and bone marrow data from females). Protein and mRNA expression data for the primary benzene-metabolizing enzymes CYP2E1 and UGT1A6 showed very little strain-dependent variation. Strain dependent differences in mRNA levels of NQO1 and MPO were small but statistically significant, as were those for GAPDH and β2-microglobulin. These data demonstrated that polymorphisms with the greatest contribution toward overall variations in systemic exposures occurred in genes encoding for non-metabolic proteins. While exposure does not equate to toxicity, identification of the genes regulating distribution and clearance may be useful for investigating host susceptibility to toxic effects following benzene exposure. This research was supported in part by the NIEHS NTP Grant N01ES45529, NIEHS Toxicology and Toxicogenomics Training Grant (5T32ES007091-29), NIEHS/NTP Division of Intramural Research, and Southwest Environmental Science Center Grant P3ES06694.

This is a research paper submitted for LIBR220 (Medical and Health Sciences Librarianship), a graduate course in Library and Information Sciences at San Jose State University. / PURPOSE: To evaluate ways in which hospital librarians can help clinicians keep up with the rapid growth of genetic information and incorporate it into patient care as we enter the era of genomic medicine. SETTING: Hospital medical libraries DESCRIPTION: The fast-growing new field of genomic medicine applies human genetic information to the understanding and treatment of disease. Historically, evidence-based healthcare has been informed by studies on large populations. Breakthroughs in genetic analysis technologies are presenting healthcare providers with new opportunities to diagnose and customize clinical treatment based on the genetic structure of individual patients. In the hospital setting, access to genomic medicine information resources, clinical guidelines, and continuing education will be critical in the near future. This research paper will explore resources and programs that hospital librarians can offer to clinicians, to serve their genomic medicine information needs and help them navigate through unfamiliar territory.

The origin of new coding sequence is a major puzzle in biology. The evolutionary pressures on new sequences are largely unknown, but structural constraints are thought to play a role. Previously, 3' untranslated region (UTR) conversion to open reading frame (ORF) was observed in Saccharomyces. We sought to identify genes that were polymorphic for stop codon position in S. cerevisiae and S. paradoxus. Using strain sequence data from the Saccharomyces Genome Resequencing Project, we found 1336 genes that had evidence of stop codon polymorphism. Of those, we found 62 genes that had evidence of addition to ancestral sequence that represented the conversion of ancestral 3' UTR to derived ORF. Two of these genes, YGL058W and YNL034W-A, are prime candidates for structural studies as they are short proteins with long additions and known structures. In future studies, they will be used to infer any structural constraints on newly evolving proteins.

All cellular processes depend on the coordinate expression of genes and their interactions. Regulatory sequences encoded in the genome stipulate the necessary instructions interpreted by sequence-specific transcription factors (TFs) to control the spatial-temporal output of gene expression. Detection of cis-regulatory signals is challenging, owing to the lack of distinguishing features such as open reading frames and an overwhelming excess of spurious to functional TF binding site matching sequences embedded within the vast non-coding regions of vertebrate genomes. From an evolutionary standpoint, functional alterations in cis-regulatory architecture are thought to be important in diversifying morphology and physiology in the evolution of vertebrates, which share a similar body plan and complement of genes. Correspondingly, recent studies have highlighted the plasticity of cis-regulatory architecture organization over evolutionary time, finding associations with examples of both diverged and conserved patterns of gene expression. These observations underscore the gap in our collective knowledge with respect to the rules by which TFs recognize and bind their targets in vivo, as well as how this process evolves in vertebrates, and serve as a motivating basis for this thesis work. To begin, I probed the extent of conservation and divergence of sequence and expression profiles across tissues of diverse vertebrate species, identifying thousands of candidate genes with conserved expression by microarray analysis. However, corresponding conservation of non-exonic and potentially regulatory sequence was lacking, suggestive of binding site turnover over evolutionary time. Next, I analyzed the sequence specificity of a wide array of mouse and yeast TFs, finding great diversity and complexity in their binding preferences, with many factors recognizing multiple distinct motifs. Furthermore, comparative analysis of orthologous TFs suggest well conserved binding specificities. I also demonstrate the likely biological relevance of sequences highly preferred by these TFs by revealing distinctive signatures in their distribution and organization within putative regulatory regions in each genome. Lastly, I have begun to explore the organization of cis-regulatory sequences active in vertebrate tissues by high-throughput sequencing of open chromatin. Together, these data help illuminate the organization and evolution of vertebrate regulatory architectures, providing a useful toolkit for the testing of new models and hypotheses.

It is a microbial world we live in: microbes outnumber other organisms by several orders of magnitude, and they have great importance for the environment. However, environmental microbes are notoriously difficult to grow in the laboratory, and using culture independent techniques is necessary to expand our view. In this thesis, I apply metagenomics and single-cell genomics to environmental samples from ancient human remains and lakes. First, I used metagenomics to learn about bacteria from a Neanderthal’s bone and the gut of Ötzi, a frozen natural mummy. Both were exploratory studies where the main question was what kind of bacteria are present. I found out that Streptomyces dominated this particular Neanderthal fossil, and the DNA lacked the damage that is often seen in ancient samples. Ötzi's gut sample was dominated by Clostridia and fungi belonging to Basidiomycota. Second, ten single-cell amplified genomes of freshwater Alphaproteobacterium LD12 and three metagenomes from Swedish lakes were sequenced. Comparative metagenomics allowed hypothesizing about which functions are important for microbe proliferation in freshwater, pointing to osmoregulation and transport proteins and, possibly, to different strategies of metabolizing sugars. I also focused on SAR11 sister-groups in oceans and lakes. Phylogenies and sequence evolutionary distance estimates indicated the existence of microclusters within LD12, showing variation in abundance between lakes. The most striking difference was the relative amount of recombination compared to mutation, the estimated r/m ratio. SAR11 marine and their freshwater cousins are found at the opposite extremes of the r/m range, lowest and highest, respectively. The genetic background or sequence diversity did not explain the observed dramatic difference, so it is possibly connected to environmental adaptation or population dynamics. In addition, I have spent a substantial amount of effort benchmarking available metagenomic methods, for example fragment recruitment of metagenomes to reference genomes. In conclusion, my exploratory metagenomic studies have shed some light on the bacteria present in ancient human remains; comparative metagenomics has suggested the importance of substrate preference on functional differences between lakes and oceans; finally, single-cell genomes have allowed some insight into molecular evolutionary processes taking place in the freshwater LD12 bacterium.

Activation-tagging is a functional genomics technique where strong enhancers are inserted randomly into target genomes to over-activate endogenous genes. Phenotypes of interest can be selected for investigation of genetic factors contributing to the mutant phenotype. From initial screens of a population of activation-tagged potato, a mutant with chocolate-coloured tuber skin has been identified. In this thesis, a novel sequence capture method for identifying T-DNA loci in activation tagged potato was used to characterize chocolate’s single T-DNA insertion locus. Transcriptome analysis of tuber periderm tissue was used to identify major processes occurring in the chocolate mutant. Our data suggest activation of a chitin-binding receptor-like kinase located 65 kb from T-DNA insert may cause activation of immune signaling pathways in chocolate. The present work explores a putative model of transcriptional and cellular responses involved in gain-of-function immune receptor activation. Selectively, these findings illustrate the periderm tissue as an important area of defense charged against biotic and abiotic stresses. Periderm development and anatomy are highly important for tuber storage. Further characterization of potato tuber periderm may contribute knowledge to model periderm systems and have implications for molecular breeding strategies to improve tuber storage quality. / Thesis (Master, Biology) -- Queen's University, 2012-09-27 11:45:16.478