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ANALYSIS OF THE MOLECULAR EVOLUTION OF THE ZOONOTIC AGENT Bartonella henselae BY SEQUENCING NINE GENES Alix Boulouis The cat-scratch disease, which is one of the most common diseases in the world transmissible from animals to humans, is caused by a bacterium, Bartonella henselae. This bacterium infects 30-60% of domestic cats in the US. It can be transmitted to humans by either a cat scratch or a cat bite. In the case of the cat scratch, the mechanism is quite easy to understand: the cat flea (Ctenocephalides felis), which is the transmission agent between cats, makes the cat scratch herself which contamines her claws. The presence of B. henselae in humans is very difficult to estimate, as the symptoms are at the same time varying and generally benign for patients with operational immune systems. B. henselae infects vessel and red blood cells and can cause cancerlike growth of the vessel cells in immunocompromised patients such as those who are HIV-positive. These bacteria can be isolated from cat and human infections, and a quite large panel of different strains is now available for laboratory experiments. The total genetic information of one of them, Houston-1, has recently been identified, and a DNA microarray experiment has been carried out on several strains to compare them with the sequenced Houston-1 genome. In the microarray experiment small pieces of DNA from the Houston-1 strain (probes) were anchored on a plate, and fragments of the genomic DNA from the strains to be analyzed were added to this: if this genomic DNA contained the same genes as were present in the array, they bound to them, which could be measured with biochemistry techniques. A microarray analysis allows one to see the differences in the gene content among the strains, and thus enables one to estimate relations between the strains: it is more probable that the same change has occurred once in the common ancestor of two strains than twice independently in both strains. It is not a good evaluation of the molecular evolution of the genomes, though. Single changes in the DNA of genes can happen during the evolution, having no influence on the functionality of the gene product, but staying witnesses of the evolution and the oldness of the strain. The measurement of single substitutions in chosen genes, by amplification of the genes and sequencing, is a good complement to the microarray for the analysis of the bacteria evolution. I carried out this sequencing experiment on nine common genes of the same strains used for the microarray. The sequencing of the nine genes allowed differentiating several versions of the genes with very few differences for each gene (except one for which there was no diversity), and the combination of the alleles for each strain allowed me to define the sequence type of the strain. I found five different sequence types among these strains. The clustering of the strains was in general in agreement with the one observed in the microarray, with the exception of one strain having good similarities with a second strain in the microarray, whereas they were different with the sequence analysis, which remains unexplained. Degree project in biology, University of Uppsala, spring 2004 Examensarbete i biologi, 10p Departments of Biology Education and Molecular Evolution, EBC, Uppsala University Supervisor: Siv Andersson