The long-term objective of the proposed research is to develop a comprehensive molecular understanding of the organism Porphyromonas gingivalis, a species strongly associated with human periodontal diseases. P. gingivalis is a small genome representative of the little studied Cytophaga/Flavobacteria/Bacteroides phylum, a phylogenetically and medically important group of organisms. The complete genome sequence of virulent strain W83, about 2.2 Mb, will be determined (Aim 1). For sequencing, a 2.0 kb average insert size library will be prepared in the pUC18 vector and an 18. kb average insert size library in lambda DashII. Approximately 35,200 sequences from both ends of 17,600 pUC clones will yield approximately 8-fold coverage of the genome. The ends of 1,000 lambda DashII clones will also be sequenced to create a genome scaffold, and to verify the orientation and integrity of the contigs formed from the assembly of sequences from the plasmid library. The genome sequence will be assembled from the collection of randomly sequenced fragments; sequencing gaps will be closed by walking appropriate clones, and physical gaps by polymerase chain reaction. The sequence will be analyzed and annotated (Aim 2). Open reading frames (Orfs) will be identified related to proteins of known and unknown function. The sequence will be analyzed to identify a number of features, including metabolic pathways, virulence factors, and exported proteins. DNA structural motifs (such as promoters, replication origins and repeat elements) and genes for rRNA, and small RNAs, such as tRNA, 4.5S, 6S, 10S, and RNAse P, will be identified. The information from the annotated sequence will be integrated with experimental data from the P. gingivalis research community, including in vivo expression technology (Aim 3). The genome sequence and annotation data will be made available on the TIGR Microbial Database web site, as well as deposited in GenBank and other appropriate databases (Aim 4). The sequence information generated in this proposal will accelerate ongoing P. gingivalis research to identify and characterize virulence factors and to develop an effective P. gingivalis vaccine. Periodontal disease affects one in five Americans, and therefore, advances in prevention and therapy will have a significant effect on the health of the American people, and the cost of dental care.
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