In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.

A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington's disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.

Though mitochondrial DNA is prone to mutation and few mtDNA repair mechanisms exist1, crippling mitochondrial mutations are exceedingly rare2. Recent studies demonstrated strong purifying selection in the mouse female germline3,4. However, the mechanisms underlying the positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila oogenesis. We found that mtDNA replication commenced prior to oocyte determination during the late germarium stage, and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA mutation, mt:CoIT300I, which displayed reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of mt:CoIT300I in heteroplasmic flies was decreased both through oogenesis and over multiple generations at the restrictive temperature. Furthermore, we determined that selection against mt:CoIT300I overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for selective amplification of healthy mtDNA, which may be evolutionarily conserved to limit transmission of deleterious mutations.

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion. INTRODUCTIONThe maintenance of proper ploidy during cell division requires both the accurate replication of chromosomes and their faithful segregation during mitosis. The physical association of sister chromatids after DNA replication, or sister chromatid cohesion, is crucial for the proper segregation of sister chromatids at anaphase and is therefore critical for genome stability. In Saccharomyces cerevisiae, cohesion is mediated by a multisubunit protein complex called cohesin that is composed of at least four proteins: Smc1p, Smc3p, Mcd1p/Scc1p, and Irr1p/Scc3p (SA1 or SA2 in mammalian cells) (Guacci et al., 1997;Michaelis et al., 1997;Toth et al., 1999). Pds5p is also required for sister chromatid cohesion and its localization to chromatin requires Mcd1p/Scc1p (Hartman et al., 2000;Panizza et al., 2000). Before the onset of anaphase, Esp1p, a protease required for the separation of sister chromatids, is bound to its inhibitor Pds1p (Ciosk et al., 1998). At the onset of anaphase Pds1p is ubiquitinated and targeted for degradation by the anaphase promoting complex/cyclosome (APC/C) (Cohen-Fix et al., 1996). Degradation of Pds1p releases Esp1p, which then cleaves the cohesin subunit Scc1p resulting in sister chromatid separation (Uhlmann et al., , 2000.Other proteins required for proper sister chromatid cohesion function during the establishment of cohesion, which takes place during S phase. Scc2p and Scc4p physically interact with each other but are not core components of the cohesin complex (Ciosk et al., 2000). Scc2p and Scc4p are, however, required for the association of cohesin with DNA (Ciosk et al., 2000). Eco1p/Ctf7p is also required for the establishment but not the maintenance of cohesion (Skibbens et al., 1999;Toth et al., 1999). In eco1 mutants, the cohesin complex is able to associate with DNA, but proper sister chromatid cohesion is not establi...

Summary It is not known how selection affects mutations on the multicopied mitochondrial genome 1–11. We transferred cytoplasm between D. melanogaster embryos carrying mitochondrial mutations to create heteroplasmic lines transmitting two mitochondrial genotypes. Increased temperature imposed selection against a temperature sensitive mutation in cytochrome oxidase, driving declines in the abundance of the mutant genome over successive generations. Selection did not influence the health or fertility of the flies, but acted during mid oogenesis to influence competition between the genomes. While mitochondria might incur an advantage by selective localization, survival or proliferation, timing and insensitivity to parkin mutation suggest that preferential proliferation underlies selection. Selection drove complete replacement of the temperature sensitive mitochondrial genome by a wild type genome, but selection also stabilized multigenerational transmission of two genomes carrying complementing detrimental mutations. While so balanced, stably transmitted detrimental mutations have no phenotype but their segregation could contribute to disease phenotypes and somatic aging.