Search

Modulating gene dose is an effective way to alter protein levels and modify phenotypes to understand gene function. In addition, combining gene-dose alleles with chemical perturbation can provide insight into drug-gene interactions. Here, we present a strategy that combines diverse loss-of-function alleles to systematically modulate gene dose in Saccharomyces cerevisiae. The generated gene dosage allele set expands the genetic toolkit for uncovering novel phenotypes.

Advances in biomarker discovery, synthetic biology and next-generation genomics promise to make designer cells a reality in the near future. Such cells can be engineered to report on diverse aspects of their physiology, potentially opening up new avenues of biological inquiry and enhancing our understanding of cell function.

Background: Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug., Results: Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches--analysis of the sensitivity of rho0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C., Conclusion: Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.

The approximately 6,000 strains in the yeast deletion collection can be studied in a single culture by using a microarray to detect the 20 bp DNA "barcodes" or "tags" contained in each strain. Barcode intensities measured by microarray are compared across time-points or across conditions to analyze the relative fitness of each strain. The development of this pooled fitness assay has greatly facilitated the functional annotation of the yeast genome by making genome-wide gene-deletion studies faster and easier, and has led to the development of high throughput methods for studying drug action in yeast. Pooled screens can be used for identifying gene functions, measuring the functional relatedness of gene pairs to group genes into pathways, identifying drug targets, and determining a drug's mechanism of action. This process involves five main steps: preparing aliquots of pooled cells, pooled growth, isolation of genomic DNA and PCR amplification of the barcodes, array hybridization, and data analysis. In addition to yeast fitness applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, siRNA vectors, and molecular inversion probes.

The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially approximately 100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical-genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure-activity relationships., Competing Interests: The authors have declared that no competing interests exist.

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.

Determining the mode of action of bioactive compounds, including natural products, is a central problem in chemical biology. Because many genes are conserved from the yeast Saccharomyces cerevisiae to humans and a number of powerful genomics tools and methodologies have been developed for this model system, yeast is making a major contribution to the field of chemical genetics. The set of barcoded yeast deletion mutants, including the set of approximately 5000 viable haploid and homozygous diploid deletion mutants and the complete set of approximately 6000 heterozygous deletion mutants, containing the set of approximately 1000 essential genes, are proving highly informative for identifying chemical-genetic interactions and deciphering compound mode of action. Gene deletions that render cells hypersensitive to a specific drug identify pathways that buffer the cell against the toxic effects of the drug and thereby provide clues about both gene and compound function. Moreover, compounds that show similar chemical-genetic profiles often perturb similar target pathways. Gene dosage can be exploited to discover connections between compounds and their targets. For example, haploinsufficiency profiling of an antifungal compound, in which the set of approximately 6000 heterozygous diploid deletion mutants are scored for hypersensitivity to a compound, may identify the target directly. Creating deletion mutant collections in other fungal species, including the major human fungal pathogen Candida albicans, will expand our chemical genomics tool set, allowing us to screen for antifungal lead drugs directly. The yeast deletion mutant collection is also being exploited to map large-scale genetic interaction data obtained from genome-wide synthetic lethal screens and the integration of this data with chemical genetic data should provide a powerful system for linking compounds to their target pathway. Extensive application of chemical genetics in yeast has the potential to develop a small molecule inhibitor for the majority of all approximately 6000 yeast genes.

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.

The yeast deletion collection includes approximately 4700 strains deleted for both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally interact with drugs. In the present study, the complete pool of approximately 4700 barcoded homozygous deletion strains of Saccharomyces cerevisiae were surveyed to identify genes/pathways interacting with 1-methyl-4-phenylpyridinium (MPP(+)) and N,N-dimethyl-4-4-bipiridinium (paraquat), neurotoxicants that can produce Parkinson's disease. Each yeast mutant is molecularly "barcoded" the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these screens allowed us to determine that the multivesicular body pathway is an important element of toxicity induced by both MPP(+) and paraquat. When yeast genes that when deleted showed sensitivity to MPP(+) and paraquat toxicity were analyzed for their homology to human genes, 80% were found to have highly conserved human homologs (with e < 10(-8)). Future work will address if these human genes may also functionally interact with MPP(+) and paraquat toxicity.

In embryos of the "modern" sea urchin species, subclass Euechinoidea, primary mesenchyme cells are derived from the progeny of micromeres formed at the sixteen cell stage of embryogenesis. The micromeres reside within the vegetal plate epithelium and later ingress into the blastocoel as primary mesenchyme cells which form the larval skeleton. Embryos of Eucidaris tribuloides, a member of the "primitive" subclass Perischoechinoidea, exhibit several noteworthy differences from euechinoid primary mesenchyme cell lineage including variable numbers and sizes of micromeres, the absence of mesenchyme ingression, and the lack of any detectable primary mesenchyme although a larval skeleton forms. In the present study, the cell lineage of the spiculogenic mesenchyme has been studied in Eucidaris tribuloides and in the euechinoid Lytechinus pictus by microinjecting the fluorescent tracer, Lucifer Yellow, into individual blastomeres of the embryo. In addition, wheat germ agglutinin, a lectin which binds only to primary mesenchyme cells of the early euechinoid embryo, was injected into the blastocoel of embryos of both species in order to examine the distribution of cells which possess primary mesenchyme-specific cell surface markers. The results of these experiments demonstrate that the spiculogenic mesenchyme of both Lytechinus and Eucidaris arise from descendants of micromeres formed at the sixteen cell stage, although the temporal and spatial distribution of these mesenchyme cells varies considerably between species. Furthermore, the evidence obtained suggests that the information necessary for spicule formation is already segregated to the vegetal pole by the eight cell stage. The results also suggest that there are no gap junctions present between the blastomeres of the early sea urchin embryo.