The megabases of DNA sequence that the genome project is amassing challenge us to discover the roles these sequences play in the life of an organism. This proposal aims to answer that challenge by using an efficient, sequence-based, genomic approach to construct a first- generation functional map of a complete eukaryotic genome. Investigations of the cell and molecular biology of Saccharomyces cerevisiae have made essential contributions to our basic understanding of how eukaryotic cells, including human cells, work. The sequence of well over half of the Saccharomyces genome has been determined, and the remainder is likely to be virtually completed within a year. Yet only about 25% of the putative genes that have been identified by sequencing the yeast genome have a function that is even partially understood. This proposal aims, in the next three years, to investigate the function of every gene in the Saccharomyces genome. Such an enterprise would be dauntingly expensive and laborious if conventional methods were used. An alternative approach, which exploits the economies of scale of a genome- wide approach, and uses the sequence as a resource, makes the proposed analysis practical and inexpensive. In the proposed """"""""genetic footprinting"""""""" method of genome-scale functional analysis, a large library of new Ty1 insertion mutations will be generated by synchronously inducing transposition in a large population of genetically-homogeneous cells. Next, representative samples of the resulting mutagenized population will each be subjected to one of a set of at least 10 diverse selective conditions. Finally, each gene will be retrospectively screened for its effects on fitness under each selective condition. The retrospective screen employs the polymerase chain reaction (PCR) to determine whether cells carrying Ty1 insertions into a subject sequence were recovered following each specific selection. A role for the specified gene in the biological activity tested by a selection can be inferred from the relative depletion (or enrichment) of the cells carrying Ty1 insertions into that gene, when cell populations harvested before and after selection are compared. Because the most expensive and laborious steps - insertional mutagenesis and selections - are carried out only once for the entire genome, rather than separately for each gene, the proposed approach to determining the functions of genes is rapid and economical compared to those in current use. The feasibility of the genetic footprinting strategy has been tested extensively. To date, nearly 90% of the 266 putative genes on Chromosome V have been successfully tested for their effects on fitness under at least 4 selective conditions. These results establish that it is feasible to produce a functional map spanning the entire yeast genome, incorporating diverse tests for each gene's function, in 3 years, at a cost of about $220 per gene.
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