HELICOBACTER PYLORI PLASMID DNA ANALYSIS

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Helicobacter pylori is a recently recognized, Gram-negative bacillus that is found adhering to the human gastric surface mucous cells. The consensus of many clinical studies is that a high frequency of association exists between both chronic type B gastritis and duodenal ulcers and colonization by H. pylori. Recent studies also find higher rates of gastric carcinoma among persons colonized with H. pylori. The exact mechanisms of H. pylori pathogenesis are not known, although many potential virulence mechanisms have been described. The majority of H. pylori studies have been clinical and/or histological observations of H. pylori interaction(s) with gastric tissue, with only a few molecular investigations reported. This project will analyze plasmid DNA isolated from H. pylori strains. No large-scale systematic analysis of H. pylori plasmids has been reported, although the PI finds that, although the plasmid molecules isolated from different clinical isolates differ in size, they do share some regions of homology. This study will investigate these regions of homology and also construct a means to begin genetic analysis of H. pylori 1) The first step will involve careful analysis of the structure of plasmid DNA isolated from a large panel of H. pylori clinical isolates. The plasmid DNA in the panel strains will be physically characterized as to size, restriction profile, and regions of DNA homology. DNA sequence analysis of homologous regions will provide information about potential polypeptides encoded and replication mechanisms of these plasmids. 2) The second step will construct a shuttle cloning vector that can be used to move H. pylori DNA between Escherichia coli and H. pylori. 3) The third step will utilize a Campylobacter-derived Kan' cassette to provide a selectable marker to determine whether any H. pylori plasmids are conjugative. In addition to providing information about the possible role of H. pylori plasmid DNA, these studies will begin the work needed to develop genetic analyses of H. pylori. As potential virulence and physiological genes are identified, a means of moving and analyzing h. pylori DNA will be important.