9304992 Segall A new method (termed the micropipet assay) has been developed to demonstrate that Saccharomyces cerevisiae cells direct growth of mating projections towards higher concentrations of mating pheromone. Current work studying yeast mating has focussed on components that are absolutely required for mating formation. The micropipet assay allows analysis of a new aspect of this pathway, gradient sensing, and thus provides the potential for identifying proteins specifically involved in this process. The work described here focuses on the signal transduction pathway that allows yeast cells to determine the direction of an external spatial gradient of mating factor. The first objective is to use this new method to assay mutants in genes potentially involved in gradient sensing. The second objective is to identify new mutants specifically defective in this orientation. The first objective will utilize the micropipet assay to characterize mutants in genes potentially involved in sensing gradients of mating factor. For example, mutants in the mating pathway, bud site selection, and cytoskeletal proteins could have altered abilities to sense and orient in spatial gradients. The second objective, identification of mutants defective in orientation in spatial gradients, will involve a two stage process. The first stage is a genetic screen for mutants with altered partner selection during mating. The phenotype expected for cells that no longer can determine the direction of spatial gradients of mating factor is a more random selection of mating partners. A simple, visual color assay of colonies on a plate will be used to identify mutants that are still able to mate, but whose choice of mating partner is no longer determined by the amount of mating factor produced by that partner. Strains that reproducibly show reduced discrimination in this assay will then be directly assayed using the micropipet assay for orientation. Strains with reduced orientati on in the micropipet assay will be grouped into complementation groups, with the eventual goal of cloning the genes via complementation with a genomic library. %%% This research is expected to provide insight into the sensing of spatial gradients of chemicals in a wide variety of organisms. ***
