A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.     

A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network (“interactome”) on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.     

Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.     

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.     

A novel phenotype of eight spores asci in deletants of the prion-like Rnq1p in Saccharomyces cerevisiae

We report that a null rnq1 mutation in the yeast RNQ1 (YCL028w) prion-like gene of so far unknown function produces the doubling of spores in the asci. This phenotype is possibly due to the lack of inhibition by Rnq1p of an additional mitotic division during ascus formation. This novel phenotype termed "octopus asci" could be similar to prion [PIN+] phenotype.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.     

A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.     

A new high resolution screening method for study of phenotype stress responses of Saccharomyces cerevisae mutants

A high resolution high throughput screening method has been developed for stress response phenotyping of the global Saccharomyces cerevisiae knock out mutant collection. Stress causing agent is added at three concentrations to individual mutant cultures growing in early exponentially phase in 384-well microplates, and the dynamic effect of stress agent exposure is measured by following subsequent growth profiles of individual mutants with a resolution of three optical density measurements per hour. Software was written for calculation of sensitivity coefficients and efficient visual inspection of the growth and inhibition curves. Three DNA damage response causing agents were chosen to explore the feasibility of the new screening method: methyl methanesulphonate, 5-fluorouracil and cisplatin. They were tested in three biological replicas on a 1400 mutant large sub-library of the homozygote diploid S. cerevisiae gene knock out collection. The sub-library consisted of only mutants with a human ortholog to the inactivated gene. Almost 400 mutants were found more sensitive to one or more of the agents. Forty-nine mutants were sensitive to all three agents. One of the mutants, ERK5, sensitive to all three agents was chosen for follow-up human cell experiments to verify that such yeast screens can be used as hypothesis generator for human cell studies. Similar to yeast, HeLa cells became more sensitive against all three DNA damaging agents when co-treated with the ERK5 inhibitor BIX21088, thus supporting the result from the yeast phenotype screen.     

The evolution of developmental timing in natural enemy systems

A population of [PSI⁺] Saccharomyces cerevisiae cells can be cured of the [PSI⁺] prion by the addition of guanidine hydrochloride (GdnHCl). In this paper we extend existing nucleated polymerisation simulation models to investigate the mechanisms that might underlie curing. Our results are consistent with the belief that prions are dispersed through the cells at division following GdnHCl addition. A key feature of the simulation model is that the probability that a polymer is transmitted from mother to daughter during cell division is dependent upon the length of the polymer. The model is able to reproduce the essential features of data from several different experimental protocols involving addition and removal of GdnHCl.     

High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae

Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets. To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge. To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae. Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves. This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult. These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant. Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress. As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells. Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics.     

Replicative aging of the yeast does not require DNA replication

Glucose addition to a stationary culture of wild-type Saccharomyces cerevisiae BY4742 cells with zero activity of MDR pumps resuspended in a fresh medium causes pump resynthesis (measured as pump-effected diS-C3(3) efflux). In a stationary culture in its original growth medium, this glucose-induced pump resynthesis fails to occur due to depletion of essential nutrients or to extracellular metabolites produced by cells during growth. Direct pump inactivation by metabolites is excluded since exponential cells with high MDR pump activity cultured in a medium with high concentration of extracellular metabolites retain this activity for at least 2 h. The metabolites also do not affect pump synthesis on the level of gene expression as addition of concentrated growth medium or an amino acid mixture to stationary cells in spent growth medium restores glucose-induced pump synthesis. The block of MDR pump synthesis is therefore due to the lack of essential nutrients in spent medium.     

5'-end formation of yeast 5.8SL rRNA is an endonucleolytic event

Like most eukaryotes, Saccharomyces cerevisiae cells contain a minor 5.8SL rRNA that, relative to the major 5.8SS species, carries several extra nucleotides at the 5'-end. The two species are produced by alternative pathways that differ in the events removing the 3'-terminal region of Internal Transcribed Spacer 1 from the 27SA2 pre-rRNA. Whereas the pathway leading to 5.8SS rRNA is well established, that producing the 5'-end of 5.8SL (called B1L) is poorly understood. Northern analysis of two different mutants of S. cerevisiae that overproduce 5.8SL rRNA revealed the presence of a fragment corresponding to the 3'-terminal region of Internal Transcribed Spacer 1 (ITS1) directly upstream from site B1L. Immunoprecipitation experiments showed this fragment to be associated with the trans-acting factor Rrp5p required for processing at the early sites A0-A3. Together these data clearly support that the 5'-end of 5.8SL rRNA is an endonucleolytic event. In vivo mutational analysis demonstrated the lack of any cis-acting sequence elements directing this cleavage within ITS1.     

Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.     

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with α-methylleucine amide as substrate within 24 h. Conversion was measured on the [M−H]⁻ species of the corresponding α-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.