Aeromonas salmonicida is a Gram negative pathogenic fish bacterium. To facilitate the construction of the chromosomal map of A.salmonicida A449, several previously uncharacterized genes, including recA and four fla genes were identified. While the location of all the genes identified as a result of this study were mapped on the chromosome, the recA and fla genes were further characterized at the molecular level. The A.salmonicida A449 recA was cloned, sequenced and expressed in vitro. The 1059 bp recA open reading frame encoded a 353 amino acid protein with predicted molecular weight (Mᵣ) of 37,900. Southern blot analysis was performed to demonstrate the high degree of conservation between the A449 recA and those of the other typical and atypical strains of A.salmonicida examined. The predicted amino acid sequence of A.salmonicida A449 RecA was found to possess a number of domains identical to those characterized in Escherichia coli RecA.These included domains for adenosine triphosphate binding, DNA binding and protein-protein interactions. The A.salmonicida A449 recA was mobilized into an E.coli recA strain and was shown to allow increased survival in the presence of the chemical mutagen methyl methane sulfonate and ultra violet (uv) irradiation. The rate of the A.salmonicida A449 recA-mediated recombination in E. coli was increased by exposure to uv light, which suggested that SOS induction in A.salmonicida paralleled that of E.coli. The A.salmonicida A449 recA also possessed a potential regulatory SOS-box in the DNA 5' of the gene. A novel flagellin operon was identified in A.salmonicida A449, characterization of which revealed the presence of two tandemly linked flagellin structural genes flaA and flaB. The flaA and flaB genes were in turn tandemly linked to flaG encoding a protein of unknown function, and flaH encoding a protein homologous to the Hook Associated Protein II of other bacteria. The flaA and flaB genes with 79% nucleotide sequence identity, were conserved in typical and atypical strains of A.salmonicida, and displayed significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA, flaB and flaG encode unprocessed proteins with predicted Ms of 32,351, 32,056 and 15,965 respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in E. coli DH5α, and FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A.salmonicida flagellins are antigenically cross-reactive with A. hydrophila TF7 flagellin(s), and evolutionally closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum.Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at extremely low frequency. Finally, the physical and genetic map of the chromosome of A. salmonicida A449 was constructed using pulsed-field gel electrophoresis and Southern blot analysis. The three restriction enzymes used in the map construction were CeuI, Pmel and PacI. The chromosome of A. salmonicida A449, with an estimated size of 4,658 ± 29.75 kb, was determined to be circular in structure. Several genes of A. salmonicida, including those which encoded proteins implicated in virulence, were localized on the chromosome map. The chromosomal locations of the recA and fla genes were also identified. The global genomic relationship between the typical and atypical strains of A. salmonicida was investigated by comparing the CeuI cleavage fingerprint of the respective genomes.The results showed that the typical strains were indeed very homogenous as had been previously reported. The atypical strains expressed extensive variation both in the number of DNA fragments obtained with CeuI and also in the digestion fingerprint. The comparison of the CeuI digestion fingerprint of atypical strains revealed a clustering of some strains which suggested that this could be a powerful taxonomic tool for better classification of the atypical group. The two A. sobria strains analyzed with CeuI were also homogenous and showed significant similarities to the A. salmonicida typical strains CeuI genomic fingerprints. In contrast, four A. hydrophila strains yielded CeuI-derived fragments which like the atypical strain varied both in number and patterns. There was also minimal observed similarities between the genome of A. hydrophila strains and the A. salmonicida strains. / Graduate

La polyarthrite rhumatoïde (PR) et la maladie coronaire (MC) sont deux maladies touchant l'adulte comportant une susceptibilité génétique et partageant plusieurs chapitres physiopathologiques. Nous avons recherché des gènes de susceptibilité à la PR en employant une approche gène-candidat dans des loci suggerés par les données d'études de criblage du genome. Nous avons mis au point une étude originale (Genescaf) d'approche des gènes impliqués dans la MC. / Rhumatoid arthritis (RA) and coronary artery disease (CAD) are two adult diseases involving some genetic susceptibility genes and sharing many physiopathogenic chapters. We tried to find some RA susceptibility genes by a candidate-gene approach located in linked loci. We performed an original study (Genescaf) to approach some CAD susceptibility genes.

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No description available.

Identification and characterization of regions influencing the precise spatial and temporal expression of genes is critical to our understanding of gene regulatory networks. Connecting transcription factors to the cis-regulatory elements that they bind and regulate remains a challenging problem in computational biology. The rapid accumulation of whole genome sequences and genome-wide expression data, and advances in alignment algorithms and motif-finding methods, provide opportunities to tackle the important task of dissecting how genes are regulated. Genes exhibiting similar expression profiles are often regulated by common transcription factors. We developed a method for identifying statistically over-represented regulatory motifs in the promoters of co-expressed genes using weight matrix models representing the specificity of known factors. Application of our methods to yeast fermenting in grape must revealed elements that play important roles in utilizing carbon sources. Extension of the method to metazoan genomes via incorporation of comparative sequence analysis facilitated identification of functionally relevant binding sites for sets of tissue-specific genes, and for genes showing similar expression in large-scale expression profiling studies. Further extensions address alternative promoters for human genes and coordinated binding of multiple transcription factors to cis-regulatory modules. Sequence conservation reveals segments of genes of potential interest, but the degree of sequence divergence among human genes and their orthologous sequences varies widely. Genes with a small number of well-distinguished, highly conserved non-coding elements proximal to the transcription start site may be well-suited for targeted laboratory promoter characterization studies. We developed a “regulatory resolution” score to prioritize lists of genes for laboratory gene regulation studies based on the conservation profile of their promoters. Additionally, genome-wide comparisons of vertebrate genomes have revealed surprisingly large numbers of highly conserved non-coding elements (HCNEs) that cluster nearby to genes associated with transcription and development. To further our understanding of the genomic organization of regulatory regions, we developed methods to identify HCNEs in insects. We find that HCNEs in insects have similar function and organization as their vertebrate counterparts. Our data suggests that microsynteny in insects has been retained to keep large arrays of HCNEs intact, forming genomic regulatory blocks that surround the key developmental genes they regulate. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The process by which information encoded m an organism's DNA is used in the synthesis of functional cell products is known as gene expression. In recent years, sequencing of RNA (RNA-seq) has emerged as the preferred technology for the simultaneous measurement of transcript sequences and their abundance. The analysis of RNA-seq data presents novel challenges and many methods have been developed for the purpose of mapping reads to genomic features and expression quantification. In the first part of my thesis I developed an R based pipeline for pre-processing, expression estimation and data quality assessment of RNA-seq datasets, which formed the basis for my subsequent work on the evolution of gene expression regulation in mammals. Since changes in gene expression levels are thought to underlie many of the phenotypic differences between species, identifying and characterising the regulatory mechanisms responsible for these changes is an important goal of molecular biology. For this, I studied the regulatory divergence of liver gene expression and of isoform usage between mouse strains. I demonstrate that gene expression diverges extensively between the strains and propose that the regulatory mechanism underlying divergent expression between two closely related mammalian species is a combination of variants that arise in cis and in trans. Isoform usage diverges to a lesser extent and appears to display a larger contribution of trans acting regulatory elements to its regulation, suggesting that isoform usage may be under different evolutionary constraints. These observations have important implications for understanding mammalian gene expression divergence and for understanding how speciation occurs.

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Le métabolisme des microorganismes est traditionnellement étudié à deux échelles: d’une part, à l’échelle locale, la description des réactions métaboliques et d’autre part, à l’échelle globale, l’étude de la physiologie de la cellule. Malgré des progrès technologiques récents facilitant les études à ces deux échelles, leur exploitation conjointe demeure complexe car le comportement physiologique de la cellule résulte de l’action coordonnée de nombreuses réactions. Les modèles mathématiques globaux du métabolisme ont toutefois récemment permis de relier ces deux échelles. Dans cette thèse, nous explorerons l’utilisation de ces modèles pour compléter la connaissance des réactions à l’aide d’une catégorie particulière de données d’échelle globale : les essentialités de gènes déterminées à partir des phénotypes de croissance de mutants de délétion. Nous nous appuierons pour cela sur la bactérie Acinetobacter baylyi ADP1. Après avoir présenté les développements effectués pour reconstruire un modèle global du métabolisme d’A. baylyi, nous montrerons que la confrontation entre phénotypes observés et phénotypes prédits permet de mettre en évidence des incohérences entre les deux échelles d’observations. Nous montrerons ensuite qu’une interprétation formelle de ces incohérences permet de corriger le modèle et d’améliorer la connaissance du métabolisme. Nous illustrerons ce propos en présentant les corrections que nous avons réalisées à l’aide de phénotypes de mutants d’A. baylyi. Enfin, dans une dernière partie, nous proposerons une méthode permettant d’automatiser la correction des incohérences causées par des erreurs d’association entre gènes et réactions. / Microbial metabolism has traditionally been investigated at two different scales: the finest involves characterizing individually each reaction occurring in the cell; the largest focuses on global cell physiology. While both scales have recently benefited from technological advances, combining them remains, however, especially complex as the global physiological behavior of a cell results from the coordinated action of a large network of reactions. Mathematical modeling approaches have yet shown recently that genome-scale metabolic models could help in linking both scales. In this thesis, we explore the use of such models to expand the knowledge of reactions with a specific type of high-level data: gene essentiality data, assessed using growth phenotypes of deletion mutants. We will use as model organism the bacterium Acinetobacter baylyi ADP1, for which a genome-wide collection of gene deletion mutants has recently been created. Following a presentation of the key steps and developments that have been required to reconstruct a global metabolic model of A. baylyi, we will show that confronting observed and predicted phenotypes highlight inconsistencies between the two scales. We will then show that a formal interpretation of these inconsistencies can guide model corrections and improvements to the knowledge of metabolism. We will illustrate this claim by presenting model corrections triggered by A. baylyi mutant phenotypes. Finally, we will introduce a method that automates the correction of inconsistencies caused by wrong associations between genes and reactions.

Massive amounts of biological data are being accumulated in science. Searching for significant meaningful information and patterns from different types of data is necessary towards gaining knowledge from these large amounts of data available to users. However, data mining techniques do not normally deal with significance. Integrating data mining techniques with standard statistical procedures provides a way for mining statistically signi- ficant, interesting information from both structured and unstructured data. In this dissertation, different algorithms for mining significant biological information from both unstructured and structured data are proposed. A weighted-density-based approach is presented for mining item data from unstructured textual representations. Different algorithms in the area of radiation hybrid mapping are developed for mining significant information from structured binary data. The proposed algorithms have different applications in the ordering problem in radiation hybrid mapping including: identifying unreliable markers, and building solid framework maps. Effectiveness of the proposed algorithms towards improving map stability is demonstrated. Map stability is determined based on resampling analysis. The proposed algorithms deal effectively and efficiently with multidimensional data and also reduce computational cost dramatically. Evaluation shows that the proposed algorithms outperform comparative methods in terms of both accuracy and computation cost.

Mycobacterium avium causes disseminated infection in humans with immunodeficiency, pulmonary infections in individuals with predisposing lung conditions (e.g., pneumoconiosis), and cervical lymphadenitis in children. Twenty-five to fifty percent of late stage AIDS patients are infected with M. avium. M. avium has been recovered from drinking water and strains from water share identical DNA fingerprints with isolates recovered from patients exposed to the water. Further, M. avium is resistant to chlorine, a disinfectant commonly used in municipal water supplies. Because of the slow growth of M. avium, measuring its susceptibility to disinfectants is laborious and reaction to a potential problem is delayed. Thus, there exists a need for a rapid test to measure the antimycobacterial disinfectant capability of chlorine containing water samples. The objective of this research was to develop a rapid and quantitative assay for the viability of mycobacteria using firefly luciferase as a reporter gene for disinfection survival studies. Derivatives of M. avium strains MD1 and A5, Mycobacterium smegmatis strain VT307 and Mycobacterium bovis BCG strain Pasteur carrying the firefly luciferase gene (pLUC10) were constructed. In pLUC10-carrying strains of M. avium strain A5 and M. smegmatis strain VT307, a direct correlation was shown between the quantity of light produced and the number of cells recovered as colony forming units. In disinfection studies of both pLUC10-carrying derivatives of M. avium strain A5 and M. smegmatis strain VT307, survival, as measured in colony forming units, correlated with survival in relative light units. Luciferase measurements appear to offer a method for rapid enumeration of mycobactericidal disinfection capacity of chlorinated water. / Master of Science