We propose a large-scale characterization of the yeast genome by examining the expression of Saccharomyces cerevisiae genes at different times during the life cycle and determining the subcellular localization of many of the encoded gene products. A transposon mutagenesis scheme will be employed to produce both beta-galactosidase fusions and epitope tag fusions. The transposon will be used to generate a collection of yeast strains in which the E. coli lacZ gene is inserted into the yeast genome at random locations. Yeast strains containing random lacZ insertions will be analyzed for beta-galactosidase production at different stages of the yeast life cycle, including vegetative growth, mating and meiosis. For those strains that carry productive fusions, the subcellular location of fusion proteins will be determined using indirect immunofluorescence. Anti-beta-galactosidase antibodies will be used to detect lacZ fusion proteins, or anti-epitope antibodies will be used to detect proteins tagged with the epitope. The identities of genes expressed at specific times in the life cycle (e.g., meiosis) or whose products localize to specific subcellular locations (e.g., microtubules) will be determined by sequencing DNA adjacent to the transposon. The information generated will be maintained in a database that will be accessible to the general scientific community. Any researcher who identifies a yeast gene will be able to determine immediately whether that gene is expressed at a specific time during the yeast life cycle and whether its gene product localizes to a specific subcellular location. This project is expected to significantly enhance the characterization of the yeast genome; the technology proposed and information gained are applicable to the study of other organisms.
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| Erdman, S; Lin, L; Malczynski, M et al. (1998) Pheromone-regulated genes required for yeast mating differentiation. J Cell Biol 140:461-83 |
| Ross-Macdonald, P; Sheehan, A; Roeder, G S et al. (1997) A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 94:190-5 |
