ISOLATION OF CDNAS FOR PROTEIN INTERACTION NETWORKS

点击翻译按钮获取中文摘要

One approach to the National Human Genome Project goal of identifying all human genes has been to isolate and sequence expressed sequences or cDNAs. The cDNAs have generally been isolated on the basis of their expression in a particular tissue, or their differential expression in one setting and not another, but not on the basis of functional characteristics. Over time, this approach will undoubtedly yield significant new insights into gene function. Initially , however, this endeavor will only provide the sequence of each new gene. A more traditional approach, ongoing for years in many laboratories, has been to identify one or a small set of genes presumed to be involved in a specific cellular process, and then to invest the necessary, and often considerable, time characterizing their function in normal and disease states. In contrast to the goals of the Genome Project., this approach identifies new genes slowly. In the project proposed here, new cDNAs will be rapidly and successively isolated using a technique that will provide information about the function of each encoded protein. Because many regulatory processes in a cell are controlled by networks of interacting proteins, new cDNAs that encode the members of these networks will be isolated based on protein-protein interactions. An efficient two-hybrid system will be used repeatedly to isolate new cDNAs that encode the members of these networks will be isolate new cDNAs based on the ability of their encoded proteins to interact with other proteins. Beginning with a small set of proteins thought to function in protein interaction networks, new cDNAs will then be used in subsequent hunts to find other new cDNAs, and this process will be continued to isolate cDNAs for all of th members of a network. The method will provide the new cDNAs in a form that can be readily characterized by sequencing, and by testing whether the encoded proteins can interact with any members of a panel of 400 known proteins using a rapid interaction mating assay. This information networks they belong to. The approach provides a way to rapidly identify large numbers of new cDNAs such as those identified in the Genome Project. The efficiency of the method proposed will allow over 1000 cDNAs to be isolated and characterized in this way by one full-time and one part-time worker. Moreover, during the initial phase of this project, the feasibility of scaling-up the method further will be explored.