Gene-products of recombinant origin have substantial commercial value for pharmaceutical and technical uses. Directed evolution approaches to generate new or improved products have successfully been applied in instances where structure-function data for target molecules are limited. Current approaches use in vitro techniques to generate diversity. These methods have a number of shortcomings due to expense and difficulty in creation of large molecular diversity libraries containing new variants. The use of a concerted in vivo process that generates large libraries of cells producing variants of the starting molecule, would result in considerable cost and effort savings. The fungus Neurospora crassa has the ability to correctly express, fold, modify and secrete active eukaryotic proteins, and the ability to "shuffle" DNA sequences in vivo by high-frequency meiotic gene conversion. The addition of a method to induce targeted mutations into an expressed heterologous gene would allow rapid screening of new variants, with no additional molecular manipulations. This proposal investigates the potential to use directed retrotransposition to introduce mutation into heterologous sequences in this fungus. Experiments to demonstrate controlled transduction of transposon-reporter chimeras between genetically marked nuclei will be performed. The long term outcome of this proposal is to generate molecular diversity in target genes, for screening of new activities and products. PROPOSED COMMERCIAL APPLICATION: This proposal tests a new method for in vivo diversification of genes by retrotransposition. This error-prone process may then be used to generate simple and robust molecular diversity libraries for screening of new variants of commercially valuable gene-products. Success will lead to a novel alternative method for directed evolution of new or improved products, allowing entry into markets for new pharmaceutical, industrial and agricultural products
