Recent Past Projects:
Genomewide identification of spliced introns using a tiling microarray
One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. Introns excised from pre-mRNA molecules by the spliceosomal machinery are released in the form of lariats, in which the 5’ end of the intron RNA is linked via a phosphodiester bond to the 2’ hydroxyl of an internal adenosine residue. The lariat must be debranched by 2’-5’ phosphodiesterase prior to their turnover. In the absence or knockdown of the debranching enzyme, these lariat RNAs accumulate. We have carried out a genomewide identification of spliced intron using a genomic tiling microarray in Saccharomyces cerevisiaes by comparison of total RNA between DBR+ and dbr1 strains. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others.
DBR homologs and DBR-mediated lariat degradation were also found in other organisms. Currently, we are working on adapting the tiling array approach for genome-wide identification of introns in Drosophila and human cells. It has been reported that knockdown of the debranching enzyme in Drosophila via RNAi can cause lariat stabilization. We also applied this approach to human cell cultures and observed a similar but modest effect. We are now testing different RNAi knockdown approaches in both organisms to improve the efficiency of lariat accumulation. Analysis of lariat accumulation in these complex organisms will not only contribute to their genome annotation, but also extend our understanding of regulated and alternative splicing in these species.
Published Results:
Zhang Z, Hesselberth JR, Fields S. Genome-wide identification of spliced introns using a tiling microarray.
Genome Res. 2007 Mar 9. download pdf, supplemental data
Recombination Studies
We are interested in turning the process of recombination to our ends, using it to facilitate gene therapy and genome engineering. To this end, we have developed a system that allows for the selection of yeast in which two overlapping parts of the selectable marker for Kanamycin (KanR) are brought together. Our system features a genomic recipient locus and a plasmid donor construct (Fig 1, new window). Recombination between these elements results in the reconstitution of a functional KanR marker when the elements are united through the mating of two yeast strains, each of which carries one of the two elements. We have made and tested a variety of donor structures (Fig 2,new window), together with different effector proteins that localize to the two DNA elements of the system.
The greatest total efficiency of conversion to the KanR phenotype was obtained with the donor construct having the most homology to the recipient locus. We are therefore using this construct to screen a random yeast genomic GAL4 library to isolate peptides that promote homologous repair. To do this, UAS GAL sequences have been cloned into the middle of the recipient locus and on the flanks of the longest donor construct we currently possess; yeast expressing peptides that promote HR when bound to the donor or recipient DNA will more frequently convert to the KanR phenotype. As genome engineering will most likely feature exogenously created linear donor DNA and may also utilize lesion-targeting endonucleases, we are employing the homing endonuclease I-SceI to introduce dsDNA breaks in both the donor and recipient DNA. While this will produce a background, we will screen serially derived libraries for clones enriched by repeated selection for the ability to promote HR.
•Clem Stanyon
Yeast Aging
The budding yeast Saccharomyces cerevisiae serves as a useful organism for studying factors that determine cellular longevity. The aging of mitotically active cells in higher eukaryotes can be modeled by the replicative life span of yeast mother cells, whereas aging of post-mitotic cells more closely resembles the chronological survival of quiescent yeast during stationary phase (Figure 1, new window). We are interested in using high-throughput technologies to identify and characterize genes that modify both aspects of cellular life span.
Replicative Aging
Measurement of yeast replicative life span requires micromanipulation of daughter cells away from mother cells following each mitotic cycle. The time-consuming nature of this assay has precluded large-scale analyses of replicative aging. In collaboration with Dr. Brian Kennedy (Department of Biochemistry, University of Washington), we have developed a method to allow semi-quantitative measurement of replicative life span based on the aging properties of a small number of cells. To date, we have determined the replicative life span phenotypes for approximately 20% (~1000 strains) of the ORF deletion collection. Completion of this analysis, in collaboration with the Kennedy lab, should take approximately 2 years.
Based on our analysis to date, we have already made several important discoveries, including the surprising finding that life span extension by calorie restriction (CR) does not require the NAD-dependent histone deacetylase, Sir2. The Sir2-indepenent nature of CR is demonstrated two ways: first, calorie restriction and overexpression of Sir2 increase life span additively, and second, CR increases life span to a greater extent in cells lacking Sir2 (and Fob1) than in wild type cells (Figure 2, new window).
Published Results:
Kaeberlein, M., Kirkland, K.T., Fields, S. and Kennedy, B.K. (2004) Sir2-independent life span extension by calorie restriction in yeast. PLoS Biology Sep;2(9):E296. download pdf
We have also determined that, contrary to a prior model proposed by Lin, Guarente, and colleagues, CR does not increase yeast life span by enhancing respiration. Yeast cells completely lacking mitochondrial DNA either have a normal life span or a dramatically shortened life span. In both cases, however, CR dramatically enhances longevity, demonstrating that respiration is not required for life span extension by CR.
Published Results:
Kaeberlein M, Hu D, Kerr EO, Tsuchiya M, Westman EA, Dang N, Fields S, Kennedy BK. Increased Life Span due to Calorie Restriction in Respiratory-Deficient Yeast. PLoS Genet. 2005 Nov 25;1(5):e69. download pdf
Of the first 564 single-gene deletion strains examined, 14 show a significant increase in replicative life span relative to the parental strain. This set includes two overlapping ORFs along with several genes that code for proteins with functions related to the nutrient responsive kinases Tor and Sch9. Of particular interest is the finding that three genes involved in ribosome biogenesis (a Tor and Sch9-regulated process) were among our set of long-lived deletion strains: REI1, RPL31A, and RPL6B. Rpl31a and Rpl6b are protein components of the large ribosomal subunit and Rei1 is a protein of unknown function that we have determined plays a role in large subunit biogenesis. This has led us to propose a model whereby CR increases life span by decreasing Tor and Sch9 activity which results in decreased ribosome biogenesis and translation (Figure 3, new window).
Published Results:
Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT, Fields S, Kennedy BK. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005 Nov 18;310(5751):1193-6. download pdf
Chronological Aging
In order to examine post-mitotic survival in a high-throughput manner, we have developed a method that allows for the simultaneous determination of chronological life span for several thousand yeast strains in a highly quantitative manner (Figure 4, new window). We have used this technology to screen the ORF deletion collection for genes whose deletion affects chronological aging. From this analysis, we have identified several genes (Table 1, new window) implicated in the TOR pathway that extend chronological life span when deleted (Figure 5, new window). The TOR proteins are highly conserved from yeast to humans and promote cellular growth in response to nutrients, especially amino acids. We have found that limitation of amino acids in the media, or pharmacological inhibition of TOR using rapamycin or methionine sulfoximine (MSX) (Figure 6, new window) can extend chronological life span, similar to deletion of TOR pathway components. Additionally, many of these interventions correlate with an increased nuclear accumulation of the stress-responsive transcription factor Msn2, and a resistance to heat and oxidative stresses. We propose a model by which decreased TOR activity up-regulates the activity of stress-response transcription factors (including Msn2) and thus promotes longevity (Figure 7, new window).
Published Results:
Powers RW 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006 Jan 15;20(2):174-84. download pdf
•Trey Powers and Matt Kaeberlein
Plasmodium Protein-Protein Interaction Project
Plasmodium falciparum is a mosquito-borne protozoan parasite responsible for the most severe form of malaria. Over 500 million people worldwide are afflicted with malaria, and each year more than one million people- most of them children - die from these infections. Despite the importance of malaria in global health, much remains to be discovered about the molecular biology of these pathogens. Of the ~5,300 proteins predicted from the P. falciparum genome sequence 60% are classified as hypothetical; this designation means that they have never been studied in Plasmodium and do not have sufficient similarity to characterized proteins in other organisms to allow functional assignments to be made. To begin to understand the functions of these novel proteins, we have identified a large number of protein-protein interactions using high-throughput yeast two-hybrid searches with protein fragments derived from genes expressed in the intraerythrocytic stages of P. falciparum. In collaboration with Prolexys Pharmaceuticals, we performed over 32,000 searches and we identified 2,846 interactions involving 1,308 proteins, which corresponds to approximately a quarter of the proteins predicted from the P. falciparum genome. We identified clusters of interacting proteins likely involved in important processes for the survival and infectivity of the parasite, such as gene regulation and host cell invasion. A large fraction of our interactions involve uncharacterized proteins and thus could lead to a new understanding of the functions of those proteins.
In addition to this, we have performed 10,000 searches with P. falciparum baits against human activation domain libraries, and 11,000 searches with P. vivax baits against P. vivax activation domain libraries. We are currently analyzing these datasets.
This work was funded in part by the Structural Genomics of Pathogenic Protozoans effort led by Wim Hol in the Department of Biochemistry.
•Doug LaCount and Marissa Vignali
Published Results:
Lacount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, Hesselberth JR, Schoenfeld LW, Ota I, Sahasrabudhe S, Kurschner C, Fields S, Hughes RE. A protein interaction network of the malaria parasite Plasmodium falciparum. Nature. 2005 Nov 3;438(7064):103-7.
A network of WW domain interactions constructed using protein microarrays
We used protein microarray technology to generate a protein interaction map for twelve of the thirteen WW domains present in proteins of the yeast Saccharomyces cerevisiae (Example figure, new window). We observed a total of 1,158 interactions with these 12 domains, most of which have not previously been described. We analyzed the representation of functional annotations within the network, identifying enrichments for proteins with vacuolar and peroxisomal localization, as well as proteins involved in cofactor biosynthesis and protein turnover. The conservation of primary sequence motifs known to be recognized by WW domains was analyzed in the context of the network, and a comparative genomics approach used to dissect the occurrence of such motifs within the dataset. We analyzed the PY (Pro-Pro-Xaa-Tyr) motif in detail, and propose a novel consensus for the motif based on its conservation among orthologs of the interacting protein. The comparative approach revealed that one of the WW domain-containing proteins has an evolutionarily conserved PY motif, possibly indicating a role for WW domain multimerization in the propagation of signals derived from WW domain binding events.
•Jay Hesselberth and John Miller
Published Results:
Hesselberth JR, Miller JP, Golob A, Stajich JE, Michaud GA, Fields S. Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins. Genome Biol. 2006 Apr 10;7(4):R30. download pdf
Yeast Membrane Protein Array
We have applied the split-ubiquitin system originally described by Johnsson and Varshavsky (1994) to investigate interactions between integral membrane proteins. In brief, one protein is fused to the N-terminal half of ubiquitin (N-Ub) and a second protein is fused to the C-terminal half of ubiquitin (C-Ub). If the membrane proteins exist in close proximity, they bring the two halves of ubiquitin back together, and endogenous ubiquitin C-terminal hydrolases recognize this “reconstituted” ubiquitin and cleave the peptide bond following the last amino acid residue of ubiquitin. In the modified system of Stagljar et al (1998) there is a transcription factor fused to this residue and, upon cleavage, the transcription factor is released from the membrane to enter the nucleus to activate reporter genes.
We have generated a collection of 705 yeast proteins that are annotated as being in an “integral membrane” environment (643 proteins) along with proteins having amino acid homology to these (62). These proteins were made both as fusions with N-Ub, and as fusions to C-Ub with the transcription factor (C-UbPLV).
We tested our transformants for the successful insertion of an in-frame ORF into our C-UbPLV by screening them for an interaction with a generic “positive control”, a N-UbI fusion protein. Of the 705 proteins generated as C-UbPLV fusions, 365 showed an interaction with this wild-type version, N-UbI, which does not require a protein-protein interaction to bind to C-Ub. This result suggests that the 365 fusions bear an insert; the insert is in frame with the C-UbPLV moiety; there are no nonsense mutations in the insert; and the fusion protein is oriented such that the COOH-terminus bearing the fusion with C-UbPLV is exposed to the cytoplasm.
These 365 integral membrane proteins were screened for interactions against the full set of 705 proteins fused to N-UbG, a mutant form of the N-Ub with an isoleucine to glycine mutation at position 13 (Johnsson and Varshavsky (1994)). The pair-wise interactions between these 365 proteins and the 705 N-Ub fusion proteins were assayed similar to the two-hybrid study of Uetz et al. (2000). A set of 1985 putative interactions between 463 NubG fusions and 270 of the C-UbPLV fusions was found. This number of interactions per protein (~8 on average) is likely high due to the false-positive rate associated with this assay (the magnitude of which is not known). These false-positives are likely to result from the high effective concentration of integral membrane proteins due to both sequestration to a two-dimensional lipid bilayer, and co-transport of membrane proteins along the stages of the secretory pathway; additionally, over-expression of the fusion proteins from an episomal plasmid with an ectopic promoter will likely promote some non-physiological interactions.
An advantage of the membrane-based yeast two-hybrid system is that interactions between proteins can be detected at the physiological site of the interaction. This allows the observation of interactions occurring between proteins of most if not all subcellular membranous compartments (Figure1, new window). However, we also observe interactions between proteins whose native localization is to distinct compartments, and the system by itself is not informative in regards to the location within the cell of the interaction. In some instances (e.g., potentially the Mst27 and Tna1 interaction in the figure) the interaction may occur in early compartments of the secretory pathway, despite the mature proteins involved having disparate ultimate destinations. In other cases the observed interaction is likely to be non-physiological, and results from mislocalization of one or both of the proteins to an inappropriate compartment due to the fusion moiety.
In order to characterize our dataset with the goal of isolating those interactions that are more probably true-positives, we collaborated with Asa Ben-Hur in William Stafford Noble's group. The approach was to use a learning algorithm, the support vector machine (SVM), to classify the interactions based on the statistics of the assay as well as other datasets from the literature (e.g., synthetic-lethality, localization studies, Gene Ontology annotations, etc.). The SVM was trained using interactions found in this study that are also identified by independent experiments as examples of "true-positives". In addition to these 34 interactions, we included 22 that are supported by one computational approach (Deane et al., 2002), and 7 by a different computational analysis (Jansen et al., 2003). These 63 interactions constitute our highest confidence interactions, and are used by the SVM to identify features of the remaining interactions that support their classification as true interactions.
An interesting outcome from multiple SVM analyses is that 138 interactions are always classified as true positives by the algorithm, and 939 are never classified as true (Figure2, new window). We will therefore examine the interactions consistently predicted to be true as well as the intermediate interactions to identify potential physiological interactions. A comparison of the features of the 138 interactions that are always classified as true with those of the 138 "worst" interactions that are never classified as true shows that the SVM selects, for most features, the values that would be expected to indicate more physiological interactions as shown in the heat map (Figure3, new window).
•John Miller and Russell Lo
Published Results:
Miller J.P., Lo R.S., Ben-Hur A, Desmarais C, Stagljar I, Stafford Noble W, Fields S. Large-scale identification of yeast integral membrane protein interactions. Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12123-12128. supplemental data, download pdf
Specific Interests of the Yeast 2-Hybrid Array Screening
We were interested in using functional genomics tools to decipher two key biological processes that are dysregulated in cancer – chromosome segregation and chromatin modifications. The focusing of genome-wide tools on specific biological processes has several benefits including: 1) bringing an unbiased approach to investigate the process; 2) discovering new players involved in a process; and 3) providing information to model the process. A further aspect of using focused functional genomics is we can come much closer to extracting information that is saturating for a particular method.
The core tool we used is the genome-wide two-hybrid array technology that has been successfully used in various projects in the lab and in collaboration with other labs. The projects that we have outlined below were of high interest.
Comprehensive two-hybrid interaction map of spindle-associated proteins:
The protein interaction information for kinetochore and associated spindle proteins is extensive but far from complete. Using the two-hybrid system, we started the comprehensive analysis of protein-protein interactions involving kinetochore and spindle proteins in collaboration with the Drubin and Barnes labs (UC Berkeley). These interactions are uncovering novel connections within the kinetochore and other cellular pathways.
Chromatin modification dependent protein interaction map:
Chromatin is subject to a variety of modifications and the specificity these modifications impart upon protein binding is still poorly understood. In collaboration with Min-Hao Kuo (Michigan State), we were working towards charting protein-protein interactions that are dependent on chromatin modifications. The combination of high-throughput techniques such as the tethered catalysis two-hybrid system and selective experiments will help elucidate the function of the various types of chromatin modifications.
Published Results:
Guo, D., Hazbun, T.R., Xu, X.-J., Ng, S.-L., Fields, S. and Kuo, M.-H. (2004) A tethered catalysis two-hybrid system to identify protein-protein interactions requiring post-translational modifications. Nature Biotechnology Jul;22(7):888-892. download pdf
Yeast Unknown ORF Project:
In a collaborative effort, the two-hybrid array technology was used in conjunction with groups affiliated with the YRC to decipher the roles of 100 essential and uncharacterized yeast genes (Hazbun et al., 2003). The integration of two-hybrid data with data from additional protein-based technologies such as co-purification and mass spectrometry, localization, and protein structure prediction enabled the functional annotation of a large fraction of these yeast genes. The parallel analysis of genes by four complementary technologies has also enhanced our understanding of the properties of the two-hybrid genome-wide array. The overlap of protein-protein interactions identified by mass spectrometry compared with two-hybrid was very low, although they both predicted similar cellular roles that agreed with localization data and protein structure prediction. The two-hybrid interactions tended to occur between proteins that were annotated in more broadly related biological processes, resulting in Go term assignments (Table 1, new window) that were lower in resolution than the mass spectrometry-based terms. This possibly reflects the tendency of two-hybrid to identify interactions with proteins in related biological processes that are not necessarily part of a core complex. For example, protein interactions identified by mass spectrometry for two unknown proteins suggested a role for inter-related complexes (Figure 1, new window) in DNA repair whereas two-hybrid interactions suggested a role in DNA repair as well as links with other biological processes such as chromosome segregation, sumoylation and ubiquitination.
Published Results:
Hazbun, T.R., Malmström, L., Anderson, S., Graczyk, B.J., Fox, B., Riffle, M., Sundin, B.A., Aranda, J.D., McDonald, W.H., Chun, C., Snydsman, B.E., Bradley, P., Muller, E.G.D., Fields, S., Baker, D., Yates, J.R. III and Davis, T.N. (2003) Assigning function to yeast proteins by integration of technologies. Molecular Cell 12:1353-1365. download pdf
•Tony Hazbun
A Yeast Screen for P. falciparum Mefloquine Resistance Genes
Mefloquine is an effective antimalarial drug. Unfortunately, resistant strains of Plasmodium falciparum are beginning to arise. The P. falciparum multi-drug resistant gene (Pfmdr1) encodes an ABC transporter that is often altered in mefloquine resistant strains and is presumed to act as a drug efflux pump. Little else is known about the parasite's mefloquine resistance mechanisms. However, mefloquine-resistant strains have been reported that contain no Pfmdr1 alterations, suggesting that additional genes are involved in mefloquine resistance. As the yeast Saccharomyces cerevisiae is sensitive to mefloquine, I have used it to screen for P. falciparum genes that can confer increased mefloquine resistance. Yeast was transformed with a P. falciparum cDNA library under the control of an S. cerevisiae galactose-inducible promoter, followed by selection on mefloquine. Several mefloquine resistance candidate genes were isolated in this screen. The four with the strongest phenotype were chosen for further analysis. These encode an uncharacterized multi-transmembrane-spanning protein, two small uncharacterized proteins, and a putative Rab GTPase activator. Each was analyzed for degree of mefloquine resistance and multidrug resistance. In addition, the mefloquine resistant P. falciparum strain W2-Mef and its sensitive parent W2 were analyzed by semi-quantitative RT-PCR to determine if any of these candidate genes is upregulated in the resistant strain. One candidate was thus regulated and it has been cloned for expression and drug testing in P. falciparum.
•Mara Jeffress
Published Results:
Jeffress M, Fields S. (2005) Identification of putative Plasmodium falciparum mefloquine resistance genes. Mol Biochem Parasitol. Feb;139(2):133-9. download pdf
Interactions of Human Toll-like Receptors
Toll-like receptor ‘sensor’ proteins are expressed in epithelia and antigen presenting cells. They are localized to the endoplasmic reticulum, the plasma membrane, and phagosome-lysosome membranes. The family of 10 human receptors, named TLR1 through TLR10, detects various microbial antigens or endogenous 'danger' signals, and subsequently triggers an ancient, highly conserved, innate immune response. TLRs are activated by ligand-induced oligomerization that recruits cytoplasmic signaling molecules to the receptors’ intracellular domains. Among the recruits are the MyD88 protein, and other adapters (TIRAP/Mal, TRIF/TICAM1, TRAM and SARM) that preferentially associate with certain activated receptors to impart some level of signaling specificity. All TLR cytoplasmic domains, as well as all identified signal adapters, harbor a canonical TIR (toll - interleukin - response) domain that mediates protein-protein interactions. All ten TLRs, as well as the tumor necrosis factor receptor and some interleukin receptors, activate the NFkB transcription factor. The proteins that impart specificity to the Toll signal transduction pathways are not fully delineated (Figure 1, new window).
Experiments in mammalian cells usually measure TLR activation by expression of an NFkB -driven reporter gene. However, any given cell may express many different receptors that use MyD88 as an adapter to activate NFkB, and of course other pathways, unrelated to the TLRs, can also activate NFkB. In contrast, the simple eukaryotic yeast S. cerevisiae does not possess endogenous TLRs or any recognizable Toll signaling pathway; therefore in yeast we can directly test protein-protein interactions without interference from endogenous proteins or other signaling pathways. We are expressing human TLR TIR domains in yeast, and using the yeast two-hybrid system to study protein interactions in the signal transduction pathway.
We screened for new proteins that bind to the receptors’ cytoplasmic domains, and have found many new interacting proteins that appear to specifically associate with certain TLR cytoplasmic domains. In particular, we have found novel and specific interactions for the closely related group of TLRs 1, 2, 6, & 10 (Table 1, new window). These are candidate proteins that may affect signaling by TLR2 heterocomplexes.
We also performed a structure-function study of the TLR-MyD88 interaction. We mapped the amino acids required for TLR association with this ‘universal’ adapter protein by swapping pieces of TLR2 into the closely related TLRs 1, 6, and 10. This creates chimaeric proteins with new (MyD88-binding) function, and is allowing us to define the exact amino acid differences between MyD88-binding and non-binding TLRs (Figure 2, new window).
The TLR-TIR domains are homologous to each other but not identical; therefore, examining protein interactions with these domains will lend insight into interaction specificity and how structure relates to function for the human TLRs.
•Victoria Brown-Kennerly and Rachel Brown.
Published Results:
Brown V, Brown RA, Ozinsky A, Hesselberth JR, Fields S. Binding specificity of Toll-like receptor cytoplasmic domains. Eur J Immunol. 2006 Mar;36(3):742-53. download pdf
Genetic Screening Using Leucine Zippers
We developed a novel type of genetic screening method to identify proteins that function in a common pathway or process. The screen takes advantage of the observation that cellular processes are often initiated when a signal or upstream event causes two or more proteins to physically interact (Figure1, new window). These are usually part of a cascade of interactions that ultimately lead to the activation of the cellular process. We tested the idea that it might be possible to artificially force these interactions to occur and activate a process in the absence of its normal signal (i.e., cause a gain-of-function phenotype).
We were artificially forcing proteins to interact by fusing them to the leucine zippers from the mammalian Fos and Jun proteins (Figure2, new window). Fos and Jun leucine zippers form a stable heterodimer that can act as a tether to bring the attached proteins into close physical proximity. Using GFP and proteins that occur in specific subcellular localizations, we have shown that Fos and Jun can cause proteins to co-localize in yeast (Figure3, new window).
If artificial tethering mimics normal protein-protein interactions and recreates an activity that normally requires an upstream signal or event, it can then be used as a method to genetically screen for unknown members of a process (Figure4, new window). We can tether every yeast protein to a known component of the pathway (i.e. by coexpressing the known protein as a fusion with the Jun leucine zipper and a library of all yeast proteins fused to Fos) and look for a phenotype associated with activation of the process under study. The process should only be activated when the normal components are tethered to the known one. Since these will be fused to the Fos leucine zipper on a plasmid they can be easily identified.
A collection of all yeast proteins fused to the Fos leucine zipper might also be a useful reagent for tagging proteins. The tag can be added simply by introducing the tag (GFP, protein A, or any other polypeptide tag) fused to the Jun leucine zipper into yeast also expressing Fos fusions.
•Mike DeVit and Meg Branson
Published Results:
Devit M, Cullen PJ, Branson M, Sprague GF Jr, Fields S. (2005) Forcing interactions as a genetic screen to identify proteins that exert a defined activity. Genome Res. Apr;15(4):560-5. download pdf
Chemical Profiling of the Yeast Deletion Collection
Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains. Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. We carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen, and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multi-chemical sensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen, and 55 specific to mefloquine. We correlate these results with data from other large-scale studies to yield novel insights into cellular function.
•Chandra Tucker
Published Results:
Tucker, C.L. and Fields, S. (2004) Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress. Comparative and Functional Genomics 5:216-224. supplemental data, download pdf
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