Epistatic interactions, manifested in the effects of mutations on the phenotypes caused by other mutations, may help uncover the functional organization of complex biological networks. Here, we studied system-level epistatic interactions by computing growth phenotypes of all single and double knockouts of 890 metabolic genes in Saccharomyces cerevisiae, using the framework of flux balance analysis. A new scale for epistasis identified a distinctive trimodal distribution of these epistatic effects, allowing gene pairs to be classified as buffering, aggravating or noninteracting. We found that the ensuing epistatic interaction network could be organized hierarchically into function-enriched modules that interact with each other 'monochromatically' (i.e., with purely aggravating or purely buffering epistatic links). This property extends the concept of epistasis from single genes to functional units and provides a new definition of biological modularity, which emphasizes interactions between, rather than within, functional modules. Our approach can be used to infer functional gene modules from purely phenotypic epistasis measurements.

Duplicate genes from the whole-genome duplication (WGD) in yeast are often dispensable--removing one copy has little or no phenotypic consequence. It is unknown, however, whether such dispensability reflects insignificance of the ancestral function or compensation from paralogs. Here, using precise competition-based measurements of the fitness cost of single and double deletions, we estimate the exposed fitness contribution of WGD duplicate genes in metabolism and bound the importance of their ancestral pre-duplication function. We find that the functional overlap between paralogs sufficiently explains the apparent dispensability of individual WGD genes. Furthermore, the lower bound on the fitness value of the ancestral function, which is estimated by the degree of synergistic epistasis, is at least as large as the average fitness cost of deleting single non-WGD genes. These results suggest that most metabolic functions encoded by WGD genes are important today and were also important at the time of duplication.

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.

Robustness is the ability of a system to maintain its function despite environmental or genetic perturbation. Genetic robustness is a key emerging property of living systems and is achieved notably by the presence of partially redundant parts that result from gene duplication. Functional overlap between paralogs allows them to compensate for each other's loss, as commonly revealed by aggravating genetic interactions. However, the molecular mechanisms linking the genotype (loss of function of a gene) to the phenotype (genetic buffering by a paralog) are still poorly understood and the molecular aspects of this compensation are rarely addressed in studies of gene duplicates. Here, we review molecular mechanisms of functional compensation between paralogous genes, many of which from studies that were not meant to study this phenomenon. We propose a standardized terminology and, depending on whether or not the molecular behavior of the intact gene is modified in response to the deletion of its paralog, we classify mechanisms of compensation into passive and active events. We further describe three non-exclusive mechanisms of active paralogous compensation for which there is evidence in the literature: changes in abundance, in localization, and in protein interactions. This review will serve as a framework for the genetic and molecular analysis of paralogous compensation, one of the universal features of genetic systems.

Duplicated genes compensate for loss of one of the paralogs by up-regulating the remaining paralog only under growth conditions in which paralog activity is required for survival.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP ؉ -glutamate dehydrogenase (NADP ؉ -GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP ؉ -GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.Two pathways for ammonium assimilation and glutamate biosynthesis have been found in a variety of organisms. The first one, described by Holzer and Schneider in 1957 (12), is mediated by NADP ϩ -glutamate dehydrogenase (NADP ϩ -GDH; EC 1.4.1.4), which catalyzes the reductive amination of 2-oxoglutarate to form glutamate. In an alternative pathway demonstrated by Tempest et al. (25), glutamate is aminated to form glutamine by glutamine synthetase (GS; EC 6.3.1.2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT; EC 1.4.1.13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several microorganisms (2,13,16,23) and in higher plants (18).In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source. Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7). Cloning of the GOGAT structural gene (GLT1) and construction of null GOGAT mutants definitively established that this yeast possesses a single NADH-GOGAT enzyme (4) and that GOGATless mutants (7) which cannot be complemented with GLT1 (unpublished results) are probably impaired in GLT1 regulation. In this paper we report the characterization of strains impaired in either GDH1, GLT1, or both. Our results show that there is a third pathway for glutamate biosynthesis, mediated by an N...

In the yeast Saccharomyces cerevisiae, two NADP؉ -dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and ␣-ketoglutarate. The GDH2-encoded NAD ؉ -dependent glutamate dehydrogenase degrades glutamate producing ammonium and ␣-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in ␣-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1-and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of ␣-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of ␣-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.