Sometimes mutations in two genes produce a phenotype that is surprising in light of each mutation's individual effects. This phenomenon, which defines genetic interaction, can reveal functional relationships between genes and pathways. For example, double mutants with surprisingly slow growth define synergistic interactions that can identify compensatory pathways or protein complexes. Recent studies have used four mathematically distinct definitions of genetic interaction (here termed Product, Additive, Log, and Min). Whether this choice holds practical consequences has not been clear, because the definitions yield identical results under some conditions. Here, we show that the choice among alternative definitions can have profound consequences. Although 52% of known synergistic genetic interactions in Saccharomyces cerevisiae were inferred according to the Min definition, we find that both Product and Log definitions (shown here to be practically equivalent) are better than Min for identifying functional relationships. Additionally, we show that the Additive and Log definitions, each commonly used in population genetics, lead to differing conclusions related to the selective advantages of sexual reproduction.epistasis ͉ fitness ͉ gene function G enetic interactions have long been studied in model organisms as a means of identifying functional relationships among genes or their corresponding gene products, with the nature of these relationships depending on the types of interactions (1-3). Additionally, the extent and nature of genetic interaction are important to theoretical explanations for the selective advantage of sexual reproduction and recombination (4-7). The study of genetic interaction has become increasingly systematic and large-scale, especially in the yeast Saccharomyces cerevisiae (6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). This provides an opportunity to examine properties of different quantitative definitions of genetic interaction and their impact on biological interpretation.A quantitative genetic interaction definition has two components: a quantitative phenotypic measure and a neutrality function that predicts the phenotype of an organism carrying two noninteracting mutations. Interaction is then defined by deviation of a double-mutant organism's phenotype from the expected neutral phenotype. A double mutant with a more extreme phenotype than expected defines a synergistic (or synthetic) interaction between the corresponding mutations (synthetic lethality, in the extreme case). Alleviating or "diminishing returns" interactions, in which the double-mutant phenotype is less severe than expected, often result when gene products operate in concert or in series within the same pathway. Alleviating interactions arise, for example, when a mutation in one gene impairs the function of a whole pathway, thereby masking the consequence of mutations in additional members of that pathway.One class of phenotype, fitness, has been central to many large-scale genetic interaction studies. Althou...

Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single-and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.

Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Phenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best-known within gene families, where an important shared function may only be revealed by mutating all family members. Here we describe the ‘Green Monster’ technology enabling the precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein (GFP) reporter gene, is subjected to repeated rounds of mating, meiosis, and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci within single cells. The Green Monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To demonstrate the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 adenosine triphosphate-binding cassette transporters within clades associated with multi-drug resistance.

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