| 1002 Generation of a Yeast Two-Hybrid Genomic Library for Treponema denticola | ||
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D.A. HOWER, R. LOPEZ, R. LUX, and W. SHI, University of California - Los Angeles, School of Dentistry, Molecular Biology Institute, USA Treponema denticola (Td) is an anaerobic spirochete involved in periodontal disease. Chemotaxis is a potentially important virulence factor for this pathogenic bacterium. Objective: The goal of this project is the generation and testing of a complete and representative Td genomic library which will be used in the yeast two-hybrid system. This library will be very useful to identify and confirm protein-protein interactions in the spirochete chemotaxis pathway, as well as other important signaling pathways. Methods: The Td genomic library was created by digesting genomic DNA and ligating genomic fragments of sizes 3kb to 0.5kb into the yeast two-hybrid pGAD "prey" vector. The genomic library was tested by digesting isolated pGAD plasmids to examine the presence and size of the genomic fragment inserts. The functionality of the genomic library was tested in the yeast two-hybrid system using established protein interactions in the chemotaxis pathway. CheW is known to interact with the chemosensory MCPs. A cheW containing pGBD "bait" vector was co-transformed into yeast with the Td genomic library to determine if MCPs could be pulled out of the library. Results: The Td genomic library is complete and representative, covering the entire genome well over 3 times. Analysis of the library revealed that most of the pGAD vectors contain genomic fragments of the expected size. Using CheW as bait in the yeast two-hybrid system, both known and putative MCPs were pulled out from the Td genomic library. Conclusion: The Td genomic library created here is complete and functional, and will be a very useful tool for studying protein-protein interactions in Treponema denticola. These studies will enable us to further elucidate the details of the spirochete chemotaxis pathway in order to better understand how Treponema denticola can penetrate gum tissue and cause periodontitis. Research supported by: Washington Dental Service. | ||
| Seq #109 - Gram-negatives 10:15 AM-11:30 AM, Thursday, 11 March 2004 Hawaii Convention Center Exhibit Hall 1-2 | ||
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