The function of many genes cannot be deduced from sequence similiarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. Phage display libraries that include all of the open reading frames (ORFs) of Treponema pallidum will be constructed. T. pallidum is the causative agent of syphilis. The complete genome sequence of this organism has recently been completed. Several features of T. pallidum make it an excellent system on which to develop and test genomic phage display technology. First, with a size of 1 million base pairs, the genome is one of the smallest known. Second, there are a total of 1041 open reading frames which makes it feasible to systematically construct libraries containing each ORF in a relatively short period of time. Finally, little is known of the biology or pathogensis of this organism because a continuous culture system is not available. This severely limits the experimental options for study of the organism. Therefore, new approaches are needed to understand gene function in T. pallidum. Two types of phage display libraries will be constructed. One type will be constructed by systematically inserting each of the 1041 ORFs of T. pallidum into a phage display vector. The second library will be constructed by shotgun cloning of randomly fragmented T. pallidum genomic DNA. The libraries will be used to identify ORFs that are involved in attachment of T. pallidum to fibronectin and host cells. In addition, the libraries will be used to identify T. pallidum DNA binding proteins and locate their binding sites. This information can be used to discover T. pallidum transcriptonal regulatory networks. The work will also serve as a model system for the use of genomic phage display as a tool for functional genomics.
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