Showing papers by "Charles Boone published in 2006"

Synthetic genetic array analysis in Saccharomyces cerevisiae.

Abstract: Synthetic lethality occurs when the combination of two mutations leads to an inviable organism. Screens for synthetic lethal genetic interactions have been used extensively to identify genes whose products buffer one another or impinge on the same essential pathway. For the yeast Saccharomyces cerevisiae, we developed a method termed Synthetic Genetic Array (SGA) analysis, which offers an efficient approach for the systematic construction of double mutants and enables a global analysis of synthetic lethal genetic interactions. In a typical SGA screen, a query mutation is crossed to an ordered array of approx 5000 viable gene deletion mutants (representing approximately 80% of all yeast genes) such that meiotic progeny harboring both mutations can be scored for fitness defects. This array-based approach automates yeast genetic analysis in general and can be easily adapted for a number of different screens, including genetic suppression, plasmid shuffling, dosage lethality, or suppression.

Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae

Abstract: The study of complex biological networks and prediction of gene function has been enabled by high-throughput (HTP) methods for detection of genetic and protein interactions. Sparse coverage in HTP datasets may, however, distort network properties and confound predictions. Although a vast number of well substantiated interactions are recorded in the scientific literature, these data have not yet been distilled into networks that enable system-level inference. We describe here a comprehensive database of genetic and protein interactions, and associated experimental evidence, for the budding yeast Saccharomyces cerevisiae, as manually curated from over 31,793 abstracts and online publications. This literature-curated (LC) dataset contains 33,311 interactions, on the order of all extant HTP datasets combined. Surprisingly, HTP protein-interaction datasets currently achieve only around 14% coverage of the interactions in the literature. The LC network nevertheless shares attributes with HTP networks, including scale-free connectivity and correlations between interactions, abundance, localization, and expression. We find that essential genes or proteins are enriched for interactions with other essential genes or proteins, suggesting that the global network may be functionally unified. This interconnectivity is supported by a substantial overlap of protein and genetic interactions in the LC dataset. We show that the LC dataset considerably improves the predictive power of network-analysis approaches. The full LC dataset is available at the BioGRID ( http://www.thebiogrid.org ) and SGD ( http://www.yeastgenome.org/ ) databases. Comprehensive datasets of biological interactions derived from the primary literature provide critical benchmarks for HTP methods, augment functional prediction, and reveal system-level attributes of biological networks.

Identifying transcription factor functions and targets by phenotypic activation.

Abstract: Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology.

The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation.

Abstract: Cellular membranes consist of lipid bilayers that typically contain a complex set of different phospholipids. In addition to providing structural support and functioning in membrane fluidity, phospholipids also play a role in cellular signaling through their ability to recruit various effector proteins. The inositol-containing phospholipids, known as phosphoinositides, are exceptionally well suited for this function, since modification of their inositol headgroup can serve to promote membrane targeting of specific effector molecules. These recruited proteins often contain domains (i.e., PH, PX, FYVE, or ENTH, etc.) that bind with high affinity to a particular phosphorylated derivative of phosphatidylinositol (PI) (22). Therefore, changes in phosphorylation of the inositol headgroup, through the action of kinases and phosphatases, can serve to activate or terminate signaling pathways initiated by different effectors. Importantly, phosphoinositides have been implicated in a number of different cellular processes, including actin organization, vesicle trafficking, and cell proliferation, demonstrating their fundamental significance in diverse areas of cell biology (33). The yeast Saccharomyces cerevisiae has been a useful system for studying the roles for phosphoinositides, since several of the PI kinases, PI phosphatases, and effectors are well conserved (28). For example, PI 3-phosphate (PI3P), whose production in yeast is mediated by the Vps34 PI 3-kinase, functions in membrane trafficking in multiple species. Similarly the role of PI 4,5-biphosphate (PI4,5P2), generated by the yeast Mss4 PI4P 5-kinase, in actin organization and endocytosis is well characterized in multiple cell types (33). However, the function of PI4P is less well understood. In yeast, two essential PI 4-kinases, Stt4 and Pik1, function to generate independent pools of PI4P that are each required for cell viability (2, 11, 41). Recently studies have indicated a role for Pik1-generated PI4P at the Golgi in secretion, while Stt4 generated PI4P at the plasma membrane appears to function in actin organization and cell integrity (1, 2, 16, 38). However, genetic studies have also suggested roles for Stt4 in phospholipid metabolism (36), a yeast cell cycle checkpoint, and mitotic exit (40). Additionally, it remains unclear whether the role for Stt4-generated PI4P in actin organization is direct or through its role as a precursor to PI4,5P2 production. In addition to phosphoinositides, sphingolipids and their precursors, sphingoid bases and ceramide, have also been shown to function in actin cytoskeleton organization and endocytosis (9). Specifically, loss of the serine palmitoyltransferase Lcb1, which catalyzes the first step in sphingolipid synthesis, results in a defect in actin polarization (43). Further study identified the sphingoid base phytosphingosine as a direct activator of Pkh1 and Pkh2, homologs of mammalian phosphoinositide dependent kinase PDK1, which are also required for actin organization (13). Additionally, overexpression of Pkc1, an effector of Pkh1 and Pkh2 that regulates actin organization, could rescue the phenotypes exhibited by loss of Lcb1 function (14). However, it remains to be shown how sphingoid base-dependent activation of Pkc1 ultimately controls actin organization. Interestingly, previous work has suggested a role for phosphoinositides in sphingolipid metabolism, suggesting that these distinct lipids may function together (4). Using cells lacking Csg2, an enzyme normally required for production of the sphingolipid mannosylinositolphosphoceramide (MIPC), a screen was performed to isolate mutations that allowed cells to grow on media containing excess calcium (4). Unlike wild-type cells, csg2Δ cells fail to grow in the presence of 100 mM calcium (5). Several suppressors were isolated and further characterized, including a mutant form of Mss4. However, the mechanism behind this suppression has not been examined further. In this study, we used synthetic genetic array (SGA) analysis to further investigate the role of Stt4, combining the stt4ts mutation with the set of 4,700 viable yeast deletion mutations. Surprisingly, we found that stt4ts cells could not tolerate perturbations in long chain fatty acid elongation, which is important for normal sphingolipid biosynthesis. Moreover, we show that the Stt4- and Mss4-mediated phosphoinositide production is required for heat shock-induced sphingolipid synthesis and that the PI4,5P2 binding proteins, Slm1 and Slm2, also function in this pathway. Loss of Slm1 and Slm2 function has been shown to result in a defect in actin organization (3), which we now demonstrate can be suppressed either through the inactivation of calcineurin, a calcium/calmodulin-dependent phosphatase, or loss of the inositol phosphosphingolipid phospholipase C Isc1, both of which alter inositolphosphoceramide (IPC) metabolism. Together, these data suggest that Slm1 and Slm2 mediate cross talk between two major lipid signaling pathways that both respond to cellular stress.

Signature-tagged mutagenesis: barcoding mutants for genome-wide screens.

Abstract: DNA signature tags (molecular barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technology have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biological processes--mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.

Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex

Abstract: Erv41p and Erv46p are integral membrane proteins conserved across species. They were originally identified as abundant constituents of COPII-coated vesicles, and form a complex which cycles between the endoplasmic reticulum and Golgi complex. Yeast strains lacking these proteins are viable but display subtle secretory phenotypes. In order to obtain information about possible biological roles of this protein complex in endoplasmic reticulum to Golgi transport, we employed the Synthetic Genetic Array approach to screen for synthetic genetic interactions with the erv46 null mutation. We identified synthetic interactions with vma12, vma21, vma22 and vps1 deletion mutations. The vma21 mutation exacerbates transport defects caused by the erv46 mutation. Unexpectedly, yeast strains lacking Vma21p fail to sort the endoplasmic reticulum to Golgi v-SNARE, Bos1p, efficiently into COPII vesicles, yet these vesicles are fully fusion competent. In addition, we set out to identify, by a biochemical approach, proteins interacting with the Erv41p-Erv46p complex. We report a strong interaction between the Erv41p-Erv46p complex and endoplasmic reticulum glucosidase II. Strains lacking a cycling Erv41p-Erv46p complex display a mild glycoprotein processing defect. Summary

Modular and Scalable Apparatus for Process Automation

Abstract: An apparatus comprises one or more functional modules, conveyers, tools, and sub-modules. A functional module comprises at least one general-purpose tool interface and a device for moving the tool interface in one or more directions. A functional module may include a rotary positioning device, which rotates the tool interface about an axis. At least one functional module is used in an apparatus, and at least one tool is attached to the tool interface of a functional module. A tool performs a specific operation on a work sample. For example, a gripping tool is used to hold and release a sample container, or a scanning tool is used to scan multiple work samples. At least one conveyer is used to transfer work sample containers from one functional module to another. In addition to functional modules, conveyers, and tools, an apparatus may include one or more sub-modules. A sub-module can be a shelf for stacking sample containers, a carousel, a wash tower for sterilizing and cleaning a tool, a centrifuge device, a shaker, an incubator, a plate sealer, etc. A functional module uses one or more tools and one or more sub-modules to complete an operation. A base is provided for installing and integrating the functional modules, conveyers, tools, and sub-modules of an apparatus. At least one Computer Processing Unit (CPU) with a Human-Machine Interface (HMI) is used to control, synchronize, and integrate the operation of functional modules, conveyers, tools, and sub-modules and to provide a Graphical User Interface (GUI) through which the user can define and run a procedure.

From worm genetic networks to complex human diseases.

Abstract: Systematic mapping of genetic interactions for Caenorhabditis elegans genes involved in signaling pathways implicated in human disease reveals a network of 350 interactions. The topology of this network resembles that mapped previously in yeast, reinforcing the idea that similar networks may underlie the genetic basis of complex human disease.

Genomic approaches for identifying DNA damage response pathways in S-cerevisiae

Abstract: DNA damage response pathways have been studied extensively in the budding yeast Saccharomyces cerevisiae, yet new genes with roles in the DNA damage response are still being identified. In this chapter we describe the use of functional genomic approaches in the identification of DNA damage response genes and pathways. These techniques take advantage of the S. cerevisiae gene deletion mutant collection, either as an ordered array or as a pool, and can be automated for high throughput.

Apparatus for process automation using pin array and actuators

Abstract: An apparatus and method for transferring plurality of samples from one sample container to another one is disclosed wherein each sample is randomly accessible and can be “cherry picked”. The disclosed method of actuation allows for using a smaller number of actuators than the number of sample transferring channels or pins and thereby simplifies the design and control of the sample transferring apparatus.

Functional genomic approaches to fungal pathogenesis, drug target validation, and antifungal drug discovery.

Abstract: Genomic studies of the nonpathogenic budding yeast Saccharomyces cerevisiae are proving to be highly applicable to antifungal drug discovery Paradigms derived from S cerevisiae studies will continue to direct C albicans research and contribute to one's understanding of fungal pathogenesis and the identification of antifungal drug targets Many of these resources and their corresponding technologies have direct applications to antifungal drug discovery, but perhaps the most relevant resource is the yeast deletion mutant set A discussion of how to exploit the knowledge of both essential genes and biological networks containing nonessential genes to identify drug targets is provided in this chapter Because chemical-genetic profiling focuses on compounds that impair cell growth, it can also be applied to natural-product extracts, which often contain only one growth-inhibitory active compound Identification of the set of essential yeast genes is one of the most important results to immediately fall out of the deletion project because it immediately defined ~1,000 candidate antifungal targets In summary, the chapter highlights the incredible power of combining systematic genetics with antifungal drug target identification In particular, the availability of a complete barcoded set of deletion mutants, including conditional alleles of essential genes, for the major fungal pathogens would permit identification of the “universal fungal essential gene set,” allow a broad application of chemical genomics in these organisms, and enable a systematic analysis of phenotypes associated with pathogenicity