High-throughput knockout screen in Schizosaccharomyces pombe identifies a novel gene required for efficient homolog disjunction during meiosis I

Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II. To identify novel proteins required for proper segregation of chromosomes during meiosis, we applied a high-throughput knockout technique to delete 87 S. pombe genes whose expression is upregulated during meiosis and analyzed the mutant phenotypes. Using this approach, we identified a new protein, Dil1, which is required to prevent meiosis I homolog non-disjunction. We show that Dil1 acts in the dynein pathway to promote oscillatory nuclear movement during meiosis.     

A screen for genes involved in the anaphase proteolytic pathway identifies tsm1(+), a novel Schizosaccharomyces pombe gene important for microtubule integrity.

The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK). Because nuc2(+) encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S. pombe mutants defective in the anaphase-promoting pathway. In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains. Two of these are alleles of cut1(+), but tsm1-512 maps to a novel genetic location. The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle. After shift of early G2 cells to 36 degrees, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation. The chromosomes, however, often fail to segregate properly. Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.     

Identification of novel Drosophila meiotic genes recovered in a P-element screen.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.     

Baculovirus-based genetic screen for antiapoptotic genes identifies a novel IAP

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. We have used this pro-apoptotic property of the p35 null virus to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. We report here the identification of Tn-IAP1, a novel member of the IAP family of cell death inhibitors. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources.     

A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces pombe

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction. We also provide evidence for a novel function of Spo4, the fission yeast ortholog of Dbf4-dependent Cdc7 kinase, in regulating the length of anaphase II spindles. In the absence of Spo4, abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus. Our observation that the spo4Δ mutant phenotype can be partially suppressed by inhibiting Cdc2-as suggests that dysregulation of the activity of this cyclin-dependent kinase may cause abnormal elongation of anaphase II spindles in spo4Δ mutant cells.     

A large-scale RNAi screen identifies functional classes of genes shaping synaptic development and maintenance

Neuronal circuit development and function require proper synapse formation and maintenance. Genetic screens are one powerful method to identify the mechanisms shaping synaptic development and stability. However, genes with essential roles in non-neural tissues may be missed in traditional loss-of-function screens. In an effort to circumvent this limitation, we used neuron-specific RNAi knock down in Drosophila and assayed the formation, growth, and maintenance of the neuromuscular junction (NMJ). We examined 1970 Drosophila genes, each of which has a conserved ortholog in mammalian genomes. Knock down of 158 genes in post-mitotic neurons led to abnormalities in the neuromuscular system, including misapposition of active zone components opposite postsynaptic glutamate receptors, synaptic terminal overgrowth and undergrowth, abnormal accumulation of synaptic material within the axon, and retraction of synaptic terminals from their postsynaptic targets. Bioinformatics analysis demonstrates that genes with overlapping annotated function are enriched within the hits for each phenotype, suggesting that the shared biological function is important for that aspect of synaptic development. For example, genes for proteasome subunits and mitotic spindle organizers are enriched among the genes whose knock down leads to defects in synaptic apposition and NMJ stability. Such genes play essential roles in all cells, however the use of tissue- and temporally-restricted RNAi indicates that the proteasome and mitotic spindle organizers participate in discrete aspects of synaptic development. In addition to identifying functional classes of genes shaping synaptic development, this screen also identifies candidate genes whose role at the synapse can be validated by traditional loss-of-function analysis. We present one such example, the dynein-interacting protein NudE, and demonstrate that it is required for proper axonal transport and synaptic maintenance. Thus, this screen has identified both functional classes of genes as well as individual candidate genes that are critical for synaptic development and will be a useful resource for subsequent mechanistic analysis of synapse formation and maintenance.     

Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like Receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi . In the present study, we have used an in vivo RNAi strategy to inactivate 75 distinct Drosophila SP genes. We then screened this collection for SPs regulating the activation of the Toll pathway by gram-positive bacteria. Here, we report the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs.     

A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae

The late endosome and vacuole of yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome. The late endosome is the convergence point of the biosynthetic and endocytic trafficking to the vacuole. Here, we describe a novel immunodetection screen to isolate mutants defective in trafficking the soluble hydrolase carboxypeptidase Y (CPY) at the late endosome to vacuole interface (env mutants). Mutants exhibit vacuolar morphology and endocytosis defects as assayed by electron, fluorescent, and nomarski microscopy. In biochemical assays, they internally accumulate p2CPY in a dense membrane compartment lacking vacuolar properties yet display normal secretion phenotypes. The results suggest vacuolar morphology and function defects that are exclusively at the late endosome/vacuole interface. env mutants define five complementation groups. The first gene of the collection to be cloned, ENV1 is allelic to VPS35 whose established function is in retrograde trafficking from late endosome to trans-Golgi network (TGN). Microscopic, biochemical, and growth analyses establish that env1 is distinct from other alleles of VPS35 in vacuolar morphology, growth characteristics, and internal accumulation of p2CPY. Our results indicate that ENV genes may define new gene functions at the late endosome to vacuole interface.     

A genome-wide screen in yeast identifies specific oxidative stress genes required for the maintenance of sub-cellular redox homeostasis

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E(GSH) was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.     

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.     

In vivo RNAi screen identifies candidate signaling genes required for collective cell migration in Drosophila ovary

Collective migration of loosely or closely associated cell groups is prevalent in animal development, physiological events, and cancer metastasis. However, our understanding of the mechanisms of collective cell migration is incomplete. Drosophila border cells provide a powerful in vivo genetic model to study collective migration and identify essential genes for this process. Using border cell-specific RNAi-silencing in Drosophila, we knocked down 360 conserved signaling transduction genes in adult flies to identify essential pathways and genes for border cell migration. We uncovered a plethora of signaling genes, a large proportion of which had not been reported for border cells, including Rack1(Receptor of activated C kinase) and brk(brinker), mad(mother against dpp), and sax(saxophone), which encode three components of TGF-β signaling. The RNAi knock down phenotype was validated by clonal analysis of Rack1 mutants. Our data suggest that inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Lastly, results from our screen not only would shed light on signaling pathways involved in collective migration during embryogenesis and organogenesis in general, but also could help our understanding for the functions of conserved human genes involved in cancer metastasis.     

Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.     

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.     

A genome-wide screen identifies yeast genes required for protection against or enhanced cytotoxicity of the antimalarial drug quinine

Quinine is used in the treatment of Plasmodium falciparum severe malaria. However, both the drug's mode of action and mechanisms of resistance are still poorly understood and subject to debate. In an effort to clarify these questions, we used the yeast Saccharomyces cerevisiae as a model for pharmacological studies with quinine. Following on a previous work that examined the yeast genomic expression program in response to quinine, we now explore a genome-wide screen for altered susceptibility to quinine using the EUROSCARF collection of yeast deletion strains. We identified 279 quinine-susceptible strains, among which 112 conferred a hyper-susceptibility phenotype. The expression of these genes, mainly involved in carbohydrate metabolism, iron uptake and ion homeostasis functions, is required for quinine resistance in yeast. Sixty-two genes whose deletion leads to increased quinine resistance were also identified in this screen, including several genes encoding ribosome protein subunits. These well-known potential drug targets in Plasmodium are associated with quinine action for the first time in this study. The suggested involvement of phosphate signaling and transport in quinine tolerance was also studied, and activation of phosphate starvation-responsive genes was observed under a mild-induced quinine stress. Finally, P. falciparum homology searches were performed for a selected group of 41 genes. Thirty-two encoded proteins possess homologs in the parasite, including subunits of a parasitic vacuolar H(+)-ATPase complex, ion and phosphate importers, and several ribosome protein subunits, suggesting that the results obtained in yeast are good candidates to be transposed and explored in a P. falciparum context.