Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.     

Histone H3 and H4 gene deletions in Saccharomyces cerevisiae

The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes. Strains with deletions of each of these loci were constructed by gene replacement techniques. Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable. In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally. However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression. The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid. The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay.     

Aberrant protein N-glycosylation impacts upon infection-related growth transitions of the haploid plant-pathogenic fungus Mycosphaerella graminicola

The ascomycete fungus Mycosphaerella graminicola is the causal agent of Septoria Tritici Blotch disease of wheat and can grow as yeast-like cells or as hyphae depending on environmental conditions. Hyphal growth is however essential for successful leaf infection. A T-DNA mutagenesis screen performed on haploid spores identified a mutant, which can undergo yeast-like growth but cannot switch to hyphal growth. For this reason the mutant was non-pathogenic towards wheat leaves. The gene affected, MgAlg2, encoded a homologue of Saccharomyces cerevisiae ScAlg2, an alpha-1,2-mannosyltransferase, which functions in the early stages of asparagine-linked protein (N-) glycosylation. Targeted gene deletion and complementation experiments confirmed that loss of MgAlg2 function prevented the developmental growth switch. MgAlg2 was able to functionally complement the S. cerevisiae ScAlg2-1 temperature sensitive growth phenotype. Spores of ΔMgAlg2 mutants were hypersensitive to the cell wall disrupting agent Calcofluor white and produced abnormally hypo-N-glycosylated proteins. Gene expression, proteome and glycoproteome analysis revealed that ΔMgAlg2 mutant spores show responses typically associated with the accumulation of mis-folded proteins. The data presented highlight key roles for protein N-glycosylation in regulating the switch to hyphal growth, possibly as a consequence of maintaining correct folding and localization of key proteins involved in this process.     

Systematic epistatic mapping of cellular processes

Genetic screens have identified many novel components of various biological processes, such as components required for cell cycle and cell division. While forward genetic screens typically generate unstructured ‘hit’ lists, genetic interaction mapping approaches can identify functional relations in a systematic fashion. Here, we discuss a recent study by our group demonstrating a two-step approach to first screen for regulators of the mitotic cell cycle, and subsequently guide hypothesis generation by using genetic interaction analysis. The screen used a high-content microscopy assay and automated image analysis to capture defects during mitotic progression and cytokinesis. Genetic interaction networks derived from process-specific features generate a snapshot of functional gene relations in those processes, which follow a temporal order during the cell cycle. This complements a recently published approach, which inferred directional genetic interactions reconstructing hierarchical relationships between genes across different phases during mitotic progression. In conclusion, this strategy leverages unbiased, genome-wide, yet highly sensitive and process-focused functional screening in cells.     

Investigation of RNA metabolism through large-scale genetic interaction profiling in yeast

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.     

Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays

Background  

Genome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images.     

Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.     

PCR-mediated one-step deletion of targeted chromosomal regions in haploid <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event. In this paper, we show that the PCD method efficiently deletes internal regions in a single transformation. Of the six chromosomal regions targeted for deletion by this method, five regions (16 to 38 kb in length) containing 10 to 19 nonessential genes were successfully eliminated at high efficiency. The one targeted region on chromosome XIII that was not deleted was subsequently found to contain sequences essential for yeast growth. While 14 individual genes in this region have been reported to be nonessential, synthetic lethal interactions may occur among these nonessential genes. Phenotypic analysis showed that four deletion strains still exhibited normal growth while possible synthetic growth defects were observed in another strain harboring a 19-gene deletion on chromosome XV. These results demonstrate that the PCD method is a useful tool for deleting genes and for analyzing their functions in defined chromosomal regions.     

Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions

Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gγ recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gγ recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 × 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wild-type haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification.     

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.     

A double‐mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions

Mitogen‐activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single‐mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double‐mutants are created from a large library of single‐mutant lines. Here we describe a new collection of 275 double‐mutant lines derived from a library of single‐mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high‐throughput double‐mutant generating pipeline using a system for growing Arabidopsis seedlings in 96‐well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double‐mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single‐mutant line. Seeds for this double‐mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double‐mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.     

The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis

The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.     

Ontogeny of melanophore patterns in haploid and diploid embryos of the frog,Bombina orientalis

Diploid tadpoles of the discoglossid frog, Bombina orientalis, possess a distinctive rectangular network of epidermal melanophores. The ontogeny of this network was examined and utilized as a model for the comparison of tissue integrity and cellular interactions in diploid and haploid embryos. During the process of network formation in diploids, a variety of melano-phore-melanophore interactions was observed. These included temporary contacts between neighboring melanophore processes, deviations of processes toward neighboring melanophores, and lateral extensions between closely situated, parallel processes originating from different cell bodies. None of these intercellular interactions were seen in haploid embryos. Haploid melanophores displayed fewer cytoplasmic extensions, appeared to be randomly oriented, and failed to establish the ordered network seen in diploid embryos. It was also discovered that, in comparison with diploid tissues, relative densities of melanophores and epithelial cells were not uniformly regulated in haploid embryos. These findings are interpreted as indicating that haploid embryos possess fundamental cell and tissue defects, and that the “haploid syndrome” is likely based on more than one or a few defective physiological functions.     

Quenching accumulation of toxic galactose-1-phosphate as a system to select disruption of protein-protein interactions in vivo

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.     

AlphaScreen selectivity assay for β-catenin/B-cell lymphoma 9 inhibitors

The aberrant formation of the β-catenin/B-cell lymphoma 9 (BCL9) protein–protein complex is the driving force for many diseases, including cancer. Crystallographic analyses demonstrate that the surface area in β-catenin for interacting with BCL9 is overlapped with that for the β-catenin/E-cadherin interaction. In this study, a robust AlphaScreen selectivity assay was developed to quantify inhibitor potency for the β-catenin/BCL9 interaction and selectivity for β-catenin/BCL9 over β-catenin/E-cadherin interactions. A pilot screen was performed to demonstrat the feasibility of this assay. This selectivity assay is highly sensitive and suitable for adaptation to high-throughput screening. The establishment of this assay lays the foundation for the discovery of selective inhibitors specific for β-catenin/BCL9 interactions.     

Mutations in the Myp1 Gene of Ustilago Maydis Attenuate Mycelial Growth and Virulence

Mating between haploid, budding cells of the dimorphic fungus Ustilago maydis results in the formation of a dikaryotic, filamentous cell type. Mating compatibility is governed by two mating-type loci called a and b; transformation of genes from these loci (e.g., a1 and b1) into a haploid strain of different mating type (e.g., a2 b2) allows filamentous growth and establishes a pathogenic cell type. Several mutants with a nonmycelial colony morphology were isolated after insertional mutagenesis of a filamentous, pathogenic haploid strain. The mutagenized region in one such mutant was recovered by plasmid rescue and employed to isolate a gene involved in conditioning the mycelial phenotype (myp1). An 1150 amino acid open reading frame is present at the myp1 locus; the predicted polypeptide is rich in serine residues and contains short regions with similarity to SH3 domain ligands. Construction of myp1 disruption and deletion mutants in haploid strains confirmed that this gene plays a role in mycelial growth and virulence.