Helicobacter pylori is a bacterial pathogen that colonizes the stomachs of most humans at some time during their lives, causes gastritis and duodenal ulcers, and is a risk factor for gastric ulcers and gastric carcinoma. The experiments proposed here are intended to help elucidate how H. pylori colonizes its host and causes disease, by searching for H. pylori genes whose expression is correlate with these processes. To prepare for the proposed experiments: we constructed a cosmid library of H. pylori genomic DNA; ordered the cosmids; identified a 67-member miniset that covers approximately 95% of the 1700 kb H. pylori chromosome; and mapped all currently known genes. Studies of other pathogens have shown the expression of many colonization- and virulence-associated genes to be affected by growth conditions. We propose to search for previously unknown H. pylori genes of this type by screening for mRNA transcripts whose levels increase or decrease in stationary vs. exponential phase, during growth in a gnotobiotic pig (the best current model for H. pylori infection), or after exposure to tissue from the pig stomach; and also to examine the expression under these conditions of H. pylori colonization-associated genes that are already known. These studies will involve: (i) preparing Southern blot hybridization filters that contain a panel of small restriction fragments generated by digestion of each clone in our miniset and that collectively cover the H. pylori chromosome, and that also fragments of known genes; (ii) extracting RNA from H. pylori grown in vitro, or in stomachs of infected pigs (collaboration with K. Eaton and S. Krakowka, Ohio State Univ); and (iii) hybridizing labelled cDNA made from this RNA to the filters. Specific genes whose expression is up- or down-regulated will be detected by changes in hybridization intensities, and sequenced efficiently using our transposon gamma delta-based """"""""pJANUS deletion factory"""""""" vector. Possible roles of the genes identified in this way will be inferred where possible, based on homologies and motifs shared with other genes. The roles of the H. pylori genes will be tested by making transposon insertion mutations, and analyzing mutant phenotypes. Transcriptional startpoints will be identified to help elucidate how the expression of these genes is regulated. The two gaps (less than or equal to 20 kb, and less than or equal to 100 kb)in our cosmid library will be closed. New colonization- or disease-associated genes found by others will be placed on our high resolution map.
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