Showing papers by "Charles Boone published in 2003"

Global Mapping of the Yeast Genetic Interaction Network: Discovering Gene and Drug Function

Abstract: budding yeast Saccharomyces cerevisiae, ~80% of the ~6000 genes are nonessential, indi- cating that many biological processes are buffered from the phenotypic consequences of genetic per- turbation. To examine these functional relation- ships we developed a method called Synthetic Genetic Array (SGA) analysis, which automates yeast genetics and enables a systematic and high- throughput construction of double mutants from an ordered array of ~4700 viable gene deletion mutants. In particular, double mutants showing reduced fitness (a synthetic sick phenotype) or lethality (a synthetic lethal phenotype) define functional relationships between genes and their corresponding pathways. We have undertaken a project to generate a synthetic genetic interaction network for the yeast cell with the expectation that it will represent a global map of functional relationships amongst most genes. We found that synthetic genetic interactions are more common than anticipated previously, with an average query gene displaying ~30 different interactions. Cluster analysis of a compendium of ~132 SGA screens revealed that genes displaying similar patterns of genetic interactions often encode proteins within the same pathway or complex; therefore, the yeast genetic interaction network predicts precise molec- ular roles of previously uncharacterized genes. Moreover, because a gene deletion mutation pro- vides a model for the effect of a compound that inhibits its corresponding gene product, our com- pendium of synthetic genetic profiles provides a key for determining the cellular targets of small molecules and drugs. Finally, the surprisingly large number of synthetic genetic interactions observed for defined mutations of inbred laboratory yeast strains suggests that digenic interactions of this type may also occur frequently amongst different alleles of genes found within the individuals of an outbred population and thus similar genetic inter- actions may underlie many of the inherited pheno- types in other organisms.

Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II.

Abstract: Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less β-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Large‐scale essential gene identification in Candida albicans and applications to antifungal drug discovery

Abstract: Summary Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387–391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.

Formins: signaling effectors for assembly and polarization of actin filaments

Abstract: Eukaryotic cells require filamentous actin to maintain their shape and for movement, growth and replication. New actin filaments are formed by the cutting of existing filaments or de novo through the action of specialized nucleators. The most highly characterized nucleator is the Arp2/3 complex, which nucleates the branched actin networks in the lamellae of migrating cells. Recently, Bni1p, which is a member of the formin family of proteins, has been shown to nucleate actin filaments in vitro. Formins are implicated in the formation of actin cables in yeast, stress fibers in tissue culture cells and cytokinesis in many cell types. Formins contain two highly conserved formin-homology domains, FH1 and FH2. The Bni1p FH2 domain is sufficient to mediate nucleation. The Bni1p FH1 domain binds profilin, an actin-monomer-binding protein that delivers actin to the growing barbed end of filaments. The Bni1p FH1-profilin interaction enhances nucleation. Formins participate in a number of signaling pathways that control the assembly of specific actin structures and bind the barbed end of actin filaments, thereby providing a cytoskeletal basis for the establishment of cell polarity.

Mechanism of Formin-Induced Nucleation of Actin Filaments

Abstract: A fragment of the yeast formin Bni1 containing the FH1FH2 domains increases the rate of filament nucleation from pure G-actin [Pruyne et al. (2002) Science 297, 612−615]. To determine the mechanism of nucleation, we compared the G-actin dependence of Bni1FH1FH2-induced polymerization with theoretical models. The data best fit a model suggesting that Bni1FH1FH2 stabilizes an actin dimer. We also show that nucleation increases with the square root of the Bni1FH1FH2 concentration. We demonstrate that this relationship is expected for any such nucleator, independent of nucleus size. The proline-rich FH1 domain binds profilin, and deletion of this domain decreases the contribution of profilin−actin to the nucleation. A role for profilin binding to the FH1 domain in filament nucleation was supported by the inability of Bni1FH1FH2 to utilize a mutant profilin, H133S profilin, with defective binding to polyproline. Bni1FH1FH2 partially inhibits barbed-end elongation, and we find that the rate constants for both p...

RAM: A Conserved Signaling Network That Regulates Ace2p Transcriptional Activity and Polarized Morphogenesis

Abstract: In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

Elg1 forms an alternative RFC complex important for DNA replication and genome integrity.

Abstract: Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5 This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53 We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response

Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae.

Abstract: We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.

Synthetic Lethal Analysis Implicates Ste20p, a p21-Activated Protein Kinase, in Polarisome Activation

Abstract: The p21-activated kinases Ste20p and Cla4p carry out undefined functions that are essential for viability during budding inSaccharomyces cerevisiae. To gain insight into the roles of Ste20p, we hav...

Interaction between a Ras and a Rho GTPase Couples Selection of a Growth Site to the Development of Cell Polarity in Yeast

Abstract: Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.

Yeast genomics and proteomics in drug discovery and target validation

Abstract: Small, cell permeable, and target-specific chemical ligands are highly valuable, not only as therapeutics but also as research tools. The synthesis, identification and characterization of these compounds is often a difficult task. The straightforward genetics of the budding yeast Saccharomyces cerevisiae, and the high degree of conservation of basic cellular processes between yeast and higher organisms makes yeast an excellent tool for drug development studies, particularly in regards to anticancer and anti-fungal drug discovery. Recent advances in yeast functional genomics and proteomics studies are changing the field of yeast research. Many of these new technologies are readily applicable to drug target identification and other aspects of drug discovery. This review will focus on current genetic, genomic, and proteomic methodologies in S. cerevisiae that have the potential to be useful in drug discovery and target validation.

ORFeomics: correcting the wiggle in worm genes.

Abstract: A new study attempts to amplify and clone all the predicted protein-encoding open reading frames (ORFs) for Caenorhabditis elegans. This analysis confirms many of the predicted genes but suggests roughly 50% of them require correction. Recombining the ORFs into a number of different expression systems can generate functional proteomics kits for characterizing protein activity and interaction networks.

Playing tag with the yeast proteome

Abstract: Two tagged proteome studies offer the most intimate and detailed view into the inner works of yeast cells to date.

CHAPTER 175 – Peptide Recognition Module Networks: Combining Phage Display with Two-Hybrid Analysis to Define Protein-Protein Interactions

Abstract: Many of the protein-protein interactions of macromolecular signaling complexes are mediated by domains that function as recognition modules to bind specific peptide sequences found in their partner, proteins. For example, SH3, WW, and EVH1 domains bind to proline-rich peptides, EH domains bind to peptides containing the NPF motif, and SH2 and FHA domains bind to peptides phophsorylated on Tyr and Thr, respectively. For particular modules within the same family, specificity is determined by critical residues in the binding partner flanking the core peptide motif. A major challenge is to construct protein-protein interaction networks in which every module within the predicted proteome of a sequenced organism is linked to its cognate partner. To address this problem, a four-step strategy is developed for the derivation of protein-protein interaction networks mediated by peptide recognition modules. As a test of this strategy, a protein interaction network for the SH3 domains of the yeast Saccharomyces cerevisiae was constructed .