The research program described in this application is focused on the development and implementation of new gene engineering methods. The methods that are being developed are particularly suited to the manipulation of modular genes, such as peptide synthetic genes. Our goal is to break the modular genes up into their component modules, and use those modules as building blocks for the assembly of novel new modular genes. To achieve this goal, we have developed two new approaches to gene engineering. Both approaches will be optimized and implemented during the funding period. The first approach involves the use of ribozymes(i.e., enzymes comprised of RNA) as tools for chimeric gene assembly. The second approach involves the use of RNA-overhang cloning (ROC) and NA-overhang cloning (DOC) to create chimeric genes. Finally, the ribozyme method will be combined with the ROC and OCmethods to create a new gene engineering system that takes full advantage of the strengths of the individual approaches. Efficient gene engineering methods are of fundamental importance for both basic and applied research on topics directly relevant to human health. For example, the gene engineering methods described here will be used to create combinatorial gene libraries that encode chimeric peptide synthetase genes. Naturally occurring peptide synthetases are known to synthesize important antibiotics, such as penicillin and vancomycin. Furthermore, the immunosuppressant cyclosporine is produced by a peptide synthetase. The chimeric peptide synthetase genes generated during the course of this project should encode hybrid enzymes that synthesize novel biologically active-molecules. A long-term goal of this project is to screen chimeric gene libraries to identify enzymes that synthesize novel compounds at could be developed as drugs.
| Donahue, William F; Turczyk, Brian M; Jarrell, Kevin A (2002) Rapid gene cloning using terminator primers and modular vectors. Nucleic Acids Res 30:e95 |
