In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

GO biological process enrichment for the 882 deletion strains below the threshold of detection by microarray in the BCprot relative to the gene universe of strains present in at least one deletion collectio

A comprehensive platform for highly multiplexed mammalian functional genetic screens

<p>Abstract</p> <p>Background</p> <p>Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.</p> <p>Results</p> <p>Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.</p> <p>Conclusion</p> <p>Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.</p

Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

Drug resistance is a consequence of how most modern medicines work. Drugs exert pressure on cells that causes death or the evolution of resistance. Indeed, highly specific drugs are rendered ineffective by a single DNA mutation. In this study, we apply the drug methotrexate, which is widely used in cancer and rheumatoid arthritis, and perform evolution experiments on Baker’s yeast to ask the different ways in which cells become drug resistant. Because of the conserved nature of biological pathways between yeast and man, our results can inform how the same mechanism may operate to render human cells resistant to treatment. Exposure of cells to small molecules and drug therapies imposes a strong selective pressure. As a result, cells rapidly acquire mutations in order to survive. These include resistant variants of the drug target as well as those that modulate drug transport and detoxification. To systematically explore how cells acquire drug resistance in an unbiased manner, rapid cost-effective approaches are required. Methotrexate, as one of the first rationally designed anticancer drugs, has served as a prototypic example of such acquired resistance. Known methotrexate resistance mechanisms include mutations that increase expression of the dihydrofolate reductase (DHFR) target as well as those that maintain function yet reduce the drug’s binding affinity. Recent evidence suggests that target-independent, epistatic mutations can also result in resistance to methotrexate. Currently, however, the relative contribution of such unlinked resistance mutations is not well understood. To address this issue, we took advantage of Saccharomyces cerevisiae as a model eukaryotic system that combined with whole-genome sequencing and a rapid screening methodology, allowed the identification of causative mutations that modulate resistance to methotrexate. We found a recurrent missense mutation in SEC21 (orthologous to human COPG1), which we confirmed in 10 de novo methotrexate-resistant strains. This sec21 allele (S96L) behaves as a recessive, gain-of-function allele, conferring methotrexate resistance that is abrogated by the presence of a wild-type copy of SEC21. These observations indicate that the Sec21p/COPI transport complex has previously uncharacterized roles in modulating methotrexate stress

Average log2ratios for all conditions tested by Bar-seq from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

For Bar-Seq data generated for each deletion collection and drug or environmental experimental condition relative to its control, average log2ratios for condition replicates are given

Supplemental figure legends, additional files and references from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

Supplemental figure legends, additional files and reference

GO enrichment of deletion strains present in the HLU signature from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

GO biological process enrichment for the 73 deletion strains present in the HLU deletion signature

Using <i>C</i>. <i>elegans</i> Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

<div><p>Background</p><p>The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode <i>Caenorhabditis elegans</i> as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner.</p><p>Methods/Principal findings</p><p>Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of <i>C</i>. <i>elegans</i> when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of <i>C</i>. <i>elegans</i>. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells.</p><p>Conclusions/Significance</p><p>The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. <i>C</i>. <i>elegans</i> as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.</p></div

Multiple mitochondrial complex II mutations confer resistance to CID 2747322.

<p>To confirm that mev-1 (<i>gk361</i>) is the cause of resistance in strain VC3631 to CID 2747322 we tested mitochondrial complex II mutant lines from the <i>C</i>. <i>elegans</i> Million Mutation Project (MMP). It was found that 7 of the MMP strains for the mitochondrial complex II were resistant towards CID 2747322 exposure, 15 of the strains had wild-type (none) resistance and 9 strains were sensitive towards exposure.</p