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At least 19 recordsLinked to original sources
PubMed2005

A systematic high-throughput screen of a yeast deletion collection for mutants defective in PHO5 regulation.

In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. One of these genes is PHO5, which encodes a secreted acid phosphatase. A phosphate-responsive signal transduction pathway (the PHO pathway) mediates this response through three central components: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. While signaling downstream of the Pho81/Pho80/Pho85 complex to PHO5 expression has been well characterized, little is known about factors acting upstream of these components. To identify missing factors involved in the PHO pathway, we carried out a high-throughput, quantitative enzymatic screen of a yeast deletion collection, searching for novel mutants defective in expression of PHO5. As a result of this study, we have identified at least nine genes that were previously not known to regulate PHO5 expression. The functional diversity of these genes suggests that the PHO pathway is networked with other important cellular signaling pathways. Among these genes, ADK1 and ADO1, encoding an adenylate kinase and an adenosine kinase, respectively, negatively regulate PHO5 expression and appear to function upstream of PHO81.

Acid Phosphatase↗
PubMed2025

A method for authenticating the fidelity of Cryptococcus neoformans knockout collections.

Gene knockout (KO) strain collections are important tools for discovery in microbiology. Cryptococcus neoformans, a human fungal pathogen, has an available genome-wide gene deletion collection that is widely used by the research community. We uncovered mix-ups in the assembly of the commercially available C. neoformans deletion collection of ~4,700 unique strains acquired by our laboratory. Evidence supporting a mix-up includes RNAseq analysis that identified transcripts for the gene listed as the KO. The mystery was soon solved as this same KO strain lacked RNA transcripts for a different KO strain gene found in the same plate position in an earlier partial KO collection, suggesting a plate swap between two KO collections. Therefore, we developed a quick PCR assay to distinguish the two KO collections based on the size differences between their nourseothricin (NAT)-resistance cassettes, confirmed by genome sequencing. Here, we report that nine of the first 15 plates of the 42-plate our KN99ɑ KO collection had been replaced with the corresponding plates from an earlier partial KO collection. We provide additional evidence that the remaining plates are correct, and the simple authentication method presented here serves as a quick check to identify similar mix-ups in the KO collections.IMPORTANCEGene KO strain collections are important tools for discovery in microbiology. The human fungal pathogen Cryptococcus neoformans has an available genome-wide deletion collection that is widely used by the research community. Here, we report that our KN99ɑ collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. Above all, this article serves as a reminder to users of the 2015 KO library collection to screen the plates before undertaking large phenotyping experiments.

Cryptococcus neoformans↗
PubMed2004

Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative.

Certain yeasts cause large-scale spoilage of preserved food materials, partly as a result of their ability to grow in the presence of the preservatives allowed in food and beverage preservation. This study used robotic methods to screen the collection of Saccharomyces cerevisiae gene deletion mutants for both increased sensitivity and increased resistance to sorbic acid, one of the most widely-used weak organic acid preservatives. In this way it sought to identify the non-essential, non-redundant activities that influence this resistance, activities that might be the potential targets of new preservation strategies. 237 mutants were identified as incapable of growth at pH 4.5 in presence of 2 mM sorbic acid, while 34 mutants exhibit even higher sorbate resistance than the wild-type parental strain. A number of oxidative stress-sensitive mutants, also mitochondrial mutants, are sorbate-sensitive. This appears to reflect the importance of sustaining a reducing intracellular environment (high reduced glutathione levels and NADH/NAD and NADPH/NADP ratios). Sorbate resistance is also very severely compromised in mutants lacking an acidified vacuole, in vacuolar protein sorting (vps) mutants, in mutants defective in ergosterol biosynthesis (erg mutants) and with several defects in actin and microtubule organization. Sorbate resistance is, however, elevated with the loss of the Yap5 transcription factor; with single losses of two B-type cyclins (Clb3p, Clb5p); and with loss of a plasma membrane calcium channel activated by endoplasmic reticulum stress (Cch1p/Mid1p).

Drug Resistance, Fungal↗
PubMed2003

A chemical genomics approach to understanding drug action.

The complete collection of yeast deletion strains represents a unique, living biological computer for understanding gene function. The molecular 'barcodes' present in each of the deletion strains allow a quantitative ranking of the importance of any gene under any experimental condition of choice. In this article, some of the recent results generated from experiments that exploit the yeast deletion collection to understand mechanisms of drug action are discussed.

Drug Evaluation, Preclinical↗
PubMed2005

Studying phospholipid metabolism using yeast systematic and chemical genetics.

Most phospholipid metabolic pathways in the budding yeast Saccharomyces cerevisiae are analogous to their mammalian counterparts. The biological tractability of yeast provides for an opportunity to rapidly determine functions of specific lipids or lipid metabolic pathways using both classical and chemical-genetic techniques. The recent generation of the yeast genome deletion collection revealed that approximately 75% of yeast genes are not essential for life. Coupling analysis of the yeast deletion collection with automation using high-throughput robotics enables yeast genetic screens to be more thorough and bypasses the requirement for library screens to identify genes of interest. Two high-throughput yeast genetic methods are described, systematic synthetic lethality and chemical genetics. Systematic synthetic lethality is based on the principle that inactivation of two genes separately has minimal effects on cell growth whereas inactivation of both genes simultaneously results in growth defects due to their shared requirement in a particular cellular process. Chemical genetics is the analysis of bioactive compounds to determine processes that regulate susceptibility to the compound under study, and provides powerful data regarding precise targets and mechanism of action that regulate action of the compound.

Automation↗
PubMed1986

Characterization of a collection of deletion mutants at the 3'-end of 16S ribosomal RNA of Escherichia coli.

Deletions were constructed in plasmid pKK3535 in the coding region for the 3'-end of E. coli 16S rRNA. The plasmid was cleaved with restriction endonuclease Hae2 under conditions favoring the production of single cut linear plasmid DNA and deletions were produced by digestion with exonuclease Bal31. Seven different deletions were isolated ranging in size from 90 to about 200 base pairs. Transcription of ribosomal DNA, processing of ribosomal RNA and incorporation of mutant rRNA into mutant particles was studied in UV-sensitive cells using a modified maxicell labeling procedure. The different mutants were missing defined features in the secondary structure of 16S rRNA and were characterized according to their stability, ability to be processed, sensitivity to colicin E3, and ability to bind ribosomal protein S1 and to interact with 50S subunits. These analyses show that the small stem and loop structure at positions 1350 to 1372 is necessary for the stability of rRNA. The deletion of the long terminal stem structure (1409-1491) in all mutant rRNAs does not block processing of the mutant rRNAs or S1 binding, although processing of the mutant rRNAs or S1 binding, although it does prevent the association of particles containing the mutant rRNA with 50S subunits.

Base Sequence↗
PubMed2006

Chemical genomic profiling for identifying intracellular targets of toxicants producing Parkinson's disease.

The yeast deletion collection includes approximately 4700 strains deleted for both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally interact with drugs. In the present study, the complete pool of approximately 4700 barcoded homozygous deletion strains of Saccharomyces cerevisiae were surveyed to identify genes/pathways interacting with 1-methyl-4-phenylpyridinium (MPP(+)) and N,N-dimethyl-4-4-bipiridinium (paraquat), neurotoxicants that can produce Parkinson's disease. Each yeast mutant is molecularly "barcoded" the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these screens allowed us to determine that the multivesicular body pathway is an important element of toxicity induced by both MPP(+) and paraquat. When yeast genes that when deleted showed sensitivity to MPP(+) and paraquat toxicity were analyzed for their homology to human genes, 80% were found to have highly conserved human homologs (with e < 10(-8)). Future work will address if these human genes may also functionally interact with MPP(+) and paraquat toxicity.

1-Methyl-4-phenylpyridinium↗
PubMed2004

Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

Animals↗
PubMed2006

Pmr1, a Golgi Ca2+/Mn2+-ATPase, is a regulator of the target of rapamycin (TOR) signaling pathway in yeast.

The rapamycin.FKBP12 complex inhibits target of rapamycin (TOR) kinase in TORC1. We screened the yeast nonessential gene deletion collection to identify mutants that conferred rapamycin resistance, and we identified PMR1, encoding the Golgi Ca2+/Mn2+ -ATPase. Deleting PMR1 in two genetic backgrounds confers rapamycin resistance. Epistasis analyses show that Pmr1 functions upstream from Npr1 and Gln-3 in opposition to Lst8, a regulator of TOR. Npr1 kinase is largely cytoplasmic, and a portion localizes to the Golgi where amino acid permeases are modified and sorted. Nuclear translocation of Gln-3 and Gln-3 reporter activity in pmr1 cells are impaired, but expression of functional Gap1 in the plasma membrane of a pmr1 strain in response to nitrogen limitation is enhanced. These two phenotypes suggest up-regulation of Npr1 function in the absence of Pmr1. Together, our results establish that Pmr1-dependent Ca2+ and/or Mn2+ ion homeostasis is necessary for TOR signaling.

Amino Acid Transport Systems↗
PubMed1996

Analysis of cis-acting elements required for replication of barley stripe mosaic virus RNAs.

The replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNA alpha and -gamma transcripts. The 5' and 3' noncoding regions were required for replication, and lack of a 5' cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNA alpha abrogated replication in trans. A 2-base change that produced a truncated alpha a protein lacking the first 16 amino acids also compromised the ability of RNA alpha to be replicated. In contrast, RNA beta transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs alpha and gamma, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between beta a and beta b was not absolutely required for replication, but small deletions within this region reduced the abundance of RNA beta by at least 10-fold. Deletions within the first 507 nt of the gamma a ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the gamma a ORF, the gamma a--gamma b intergenic region, and the gamma b ORF could be amplified in trans. Two mutants containing extensive deletions encompassing the central region of the gamma a ORF and most of gamma b behaved like defective interfering RNAs because they multiplied to high levels in trans and caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs alpha and gamma.

Base Sequence↗
PubMed2026

Efficient homologous replacement and deletion of large genomic fragments through template-jumping prime editing in rice.

Homologous replacement of genomic sequences with large DNA fragments (>&#x2009;100&#x2009;bp) holds great potential for crop breeding, yet an efficient method to achieve such edits is lacking in plants. Here, in rice, we developed template-jumping prime editing (TJ-PE), a recently reported PE strategy for large targeted insertion, as an efficient tool for homologous replacement with DNA fragments ranging from dozens to hundreds of base pairs, and using TJ-PE, we replaced genomic fragments of up to 340&#x2009;bp with homologous fragments of the same length. In addition, our TJ-PE tool also enabled precise deletion of 944- to 2024-bp fragments in rice, with efficiencies of up to 34.6% for c. 2000-bp precise deletions. Collectively, this study expands the editing scope of PE in rice and establishes TJ-PE as a generalist tool for precise deletion and replacement of large DNA fragments.

Oryza↗
PubMed2005

Genome-wide screen for inner nuclear membrane protein targeting in Saccharomyces cerevisiae: roles for N-acetylation and an integral membrane protein.

Appropriate nuclear membrane structure is important for all eukaryotic organisms as evidenced by the numerous human diseases and alterations in gene expression caused by inappropriate targeting of proteins to the inner nuclear membrane (INM). We report here the first genome-wide screen to identify proteins functioning in INM targeting. We transformed to near completion the 4850 members of the Saccharomyces cerevisiae deletion collection of unessential genes in the 96-well format with a plasmid encoding a reporter protein, Trm1-II-GFP, which normally resides at the INM. We found that deletion of genes encoding subunits of the N-terminal acetyltransferase, NatC, cause mislocation of Trm1-II-GFP from the INM to the nucleoplasm. Mass spectroscopic analysis indicates that Trm1-II-GFP is N-acetylated. N-terminal mutations of Trm1-II-GFP predicted to ablate N-acetylation cause nucleoplasmic location, whereas a variant with an N-terminal alteration predicted to allow N-acetylation by NatC is located at the INM, providing genetic support that Trm1p-II N-acetylation is necessary for its subnuclear INM location. However, because N-acetylation appears not to be sufficient for INM targeting, it may provide a necessary role for INM targeting by affecting Trm1-II-GFP structure and exposure of cis-acting INM targeting motifs. We also discovered that YIL090W/Ice2p, an integral membrane protein located in the endoplasmic reticulum, is necessary for efficient targeting of Trm1-II-GFP to the INM. YIL090W/Ice2p may serve as a tether for INM proteins or as a regulator of INM tethers. Our methodology can be extrapolated to obtain genome-wide perspectives of mechanisms necessary to achieve appropriate subcellular and/or suborganellar location for any resident protein.

Acetylation↗
PubMed1990

Bacteriophage Mu sites and functions involved in the inhibition of lambda::mini-Mu growth.

To better understand the nature of the mini-Mu-directed process which results in inhibition of lambda::mini-Mu growth we characterized spontaneous deletion mutants of the lambda::mini-Mu phage. On the basis of analysis of the deletion endpoints, mini-Mu replication functions, and integration and inhibition properties, the lambda::mini-Mu deletion mutants were divided into five classes which define the Mu sites and functions involved in lambda::mini-Mu growth inhibition. Class 1 mutants, which still exhibit lambda::mini-Mu growth inhibition, collectively delete all the Mu late functions encoded by the mini-Mu. Class 2 and 5 mutants, which show cis-dominant defects in inhibition and integration, delete the right and left mini-Mu attachment sites, respectively. Phages of Classes 3 and 4, which delete the Mu B or A and B genes, respectively, show recessive defects in growth inhibition. The properties of these mutants define the Mu replication functions, A and B, and the Mu attachment sites as essential for the inhibition of lambda::mini-Mu growth. The observation that the sites and functions essential for Mu replication also have requisite roles in the inhibition of lambda::mini-Mu growth suggests that inhibition results from mini-Mu-promoted replicative interference of lambda::mini-Mu development.

Bacteriophage lambda↗
PubMed1996

Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repeats 1-6 are involved in hormone binding.

Previous studies have demonstrated that the amino-terminal extracellular domain of the lutropin/choriogonadotropin receptor (LHR) is sufficient for conferring high affinity binding and binding specificity. The present study was undertaken to further delineate those regions involved in hormone binding. Since the extracellular domains of the gonadotropin receptors are defined by multiple leucine-rich repeat motifs, LHR deletion mutants were constructed in which individual or multiple leucine-rich repeats were deleted from the full-length receptor. These mutants were transiently expressed in mammalian 293 cells, assayed for protein expression by Western blotting of cell extracts, and then tested for hormone binding in both intact cells and detergent-solubilized cell extracts. Western blot analyses confirmed the expression of all LHR deletion mutant proteins in the transfected cells. Although human (h) CG binding activity was not detected for any of the mutants when intact cells were assayed, mutants in which leucine-rich repeats 9-14 were collectively deleted, or repeats 7 or 8 were individually deleted, expressed binding activity when the cells were first solubilized in detergent. Of significance, rat (r) LHR(delta LRR 9-14) exhibited high affinity hCG and hLH binding, comparable to wild type rLHR. Detergent extracts from cells expressing rLHR(delta LRR 8) and rLHR(delta LRR 7) bound hCG and hLH, albeit with reduced affinities compared with the wild type receptor. All three of these deletion mutants, rLHR(delta LRR 9-14), rLHR(delta LRR 8), and rLHR(delta LRR 7), exhibited normal binding specificity as they did not bind hFSH even at very high concentrations. Thus, deletion of these regions in the carboxyl portion of the extracellular domain of the LHR did not remove any potentially inhibitory elements that would normally prevent hFSH binding. Deletion of either the 11 amino-terminal acids before LRR 1 or LRRs 1, 2, 3, 4, 5, or 6 individually from the full-length rLHR resulted in the total absence of detectable hCG binding activity in detergent-solubilized extracts, in spite of stable protein expression. These results suggest that the amino terminus and LRRs 1-6 are absolutely essential for gonadotropin binding to the rLHR.

Binding Sites↗
PubMed2006

Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies.

Multigenic influences are major contributors to human genetic disorders. Since humans are highly polymorphic, there are a high number of possible detrimental, multiallelic gene pairs. The actin cytoskeleton of yeast was used to determine the potential for deleterious bigenic interactions; approximately 4800 complex hemizygote strains were constructed between an actin-null allele and the nonessential gene deletion collection. We found 208 genes that have deleterious complex haploinsufficient (CHI) interactions with actin. This set is enriched for genes with gene ontology terms shared with actin, including several actin-binding protein genes, and nearly half of the CHI genes have defects in actin organization when deleted. Interactions were frequently seen with genes for multiple components of a complex or with genes involved in the same function. For example, many of the genes for the large ribosomal subunit (RPLs) were CHI with act1Delta and had actin organization defects when deleted. This was generally true of only one RPL paralog of apparently duplicate genes, suggesting functional specialization between ribosomal genes. In many cases, CHI interactions could be attributed to localized defects on the actin protein. Spatial congruence in these data suggest that the loss of binding to specific actin-binding proteins causes subsets of CHI interactions.

Actins↗
PubMed2005

PROPHECY--a database for high-resolution phenomics.

The rapid recent evolution of the field phenomics--the genome-wide study of gene dispensability by quantitative analysis of phenotypes--has resulted in an increasing demand for new data analysis and visualization tools. Following the introduction of a novel approach for precise, genome-wide quantification of gene dispensability in Saccharomyces cerevisiae we here announce a public resource for mining, filtering and visualizing phenotypic data--the PROPHECY database. PROPHECY is designed to allow easy and flexible access to physiologically relevant quantitative data for the growth behaviour of mutant strains in the yeast deletion collection during conditions of environmental challenges. PROPHECY is publicly accessible at http://prophecy.lundberg.gu.se.

Computer Graphics↗