Biochemistry, Genetics and Molecular Biology › Molecular Biology

Fungal and yeast genetics research

Works Count: 48214

Wikipedia

Description

This cluster of papers focuses on the genomic expression programs, protein production, and cellular responses of yeast organisms to environmental changes, stress, and fungal pathogens. It also explores the genetic and metabolic regulation, gene knockout techniques, and signaling pathways in yeast.

Keywords

Yeast; Genomics; Gene Expression; Cell Wall; Stress Response; Fungal Pathogens; Protein Production; Signal Transduction; Environmental Changes; Metabolic Regulation

Getting started with yeast

2002-01-01

Fred Sherman

Methods in yeast genetics

1979-01-01

Fred Sherman , Gerald R. Fink , James Hicks

Signal Transduction through MAP Kinase Cascades

1998-01-01

Timothy S. Lewis , Paul Shapiro , Natalie G. Ahn

A generic protein purification method for protein complex characterization and proteome exploration

1999-10-01

Guillaume Rigaut , Anna Shevchenko , Berthold Rutz +3 more

Methods in Yeast Genetics

1988-09-01

Heiner Jacob

Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual

2005-01-01

David C. Amberg , Daniel J. Burke , Jeffrey N. Strathern

Experiments: looking at yeast cells isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants meiotic mapping mitotic recombination and random spore analysis transformation of yeast synthetic lethal mutants gene replacement … Experiments: looking at yeast cells isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants meiotic mapping mitotic recombination and random spore analysis transformation of yeast synthetic lethal mutants gene replacement isolation of ras2 suppressors manipulating cell types isolating mutants by insertional mutagenesis two-hybrid protein interaction method. Techniques and protocols: high-efficiency transformation of yeast Lazy Bones plasmid transformation of yeast colonies yeast DNA isolations yeast DNA miniprep (40 ml) yeast DNA miniprep (5 ml) a ten-minute DNA preparation from yeast yeast genomic DNA - glass bead preparation yeast protein extracts yeast RNA isolation hydroxylamine mutagenesis of plasmid DNA assay of beta-galactosidase in yeast plate assay for carboxypeptidase Y random spore analysis yeast vital stains yeast immunofluorescence actin staining in fixed cells PCR protocol for PCR-mediated gene disruption yeast colony PCR protocol measuring yeast cell density by spectrophotometry cell synchrony chromatin immunoprecipitation flow cytometry of yeast DNA logarithmic growth EMS mutagenesis tetrad dissection making a tetrad dissection needle picking zygotes determining plating efficiency. Appendices: media stock preservation yeast genetic and physical maps templates for making streak plates electrophoretic karyotypes of strains for southern blot mapping strains counting yeast cells with a standard hemocytometer chamber tetrad scoring sheet trademarks suppliers.

Molecular evidence for an ancient duplication of the entire yeast genome

1997-06-12

Kenneth H. Wolfe , Denis C. Shields

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The Genetics of Aspergillus nidulans

1953-01-01

G. Pontecorvo , J. A. Roper , L.M. Chemmons +2 more

A new efficient gene disruption cassette for repeated use in budding yeast

1996-07-01

Ulrich Güldener

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the … The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre- lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

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Mitogen-Activated Protein Kinases: Specific Messages from Ubiquitous Messengers

1999-04-01

H. J. Schaeffer , Michael J. Weber

Signal transduction networks allow cells to perceive changes in the extracellular environment and to mount an appropriate response. Mitogen-activated protein kinase (MAPK) cascades are among the most thoroughly studied of … Signal transduction networks allow cells to perceive changes in the extracellular environment and to mount an appropriate response. Mitogen-activated protein kinase (MAPK) cascades are among the most thoroughly studied of signal transduction systems and have been shown to participate in a diverse array of cellular programs, including cell differentiation, cell movement, cell division, and cell death. A key question in studies of this cascade is, how does a ubiquitously activated regulatory enzume generate a specific and biologically appropriate cellular response? In this review we describe recent findings that provide insight into ways that the regulation, structure, and localization of MAPKs and the participation of adapters and scaffolds can help determine biological outcomes. MAPK cascades are evolutionarily conserved in all eucaryotes and play a key role in the regulation of gene expression as well as cytoplasmic activities. They typically are organized in a three-kinase architecture consisting of a MAPK, a MAPK activator (MEK, MKK, or MAPK kinase), and a MEK activator (MEK kinase [MEKK] or MAPK kinase kinase). Transmission of signals is achieved by sequential phosphorylation and activation of the components specific to a respective cascade. In the yeast Saccharomyces cerevisiae, five MAPK modules have been described; they regulate mating, filamentation, high-osmolarity responses, cell wall remodeling, and sporulation (Fig. (Fig.1A)1A) (reviewed in references 56 and 77). In mammalian systems five distinguishable MAPK modules have been identified so far (Fig. (Fig.1B).1B). These include the extracellular signal-regulated kinase 1 and 2 (ERK1/2) cascade, which preferentially regulates cell growth and differentiation, as well as the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades, which function mainly in stress responses like inflammation and apoptosis (reviewed in references 57, 74, and 103). Moreover, MAPK pathways control several developmental programs, such as morphogenesis and spatial patterning in Dictyostelium amoebae (17, 45), eye development in Drosophila melanogaster (124), vulva induction in Caenorhabditis elegans (113), and T-cell development in mammals (31). FIG. 1 Schematic overview of MAPK modules. (A) In S. cerevisiae, five MAPK modules regulate mating, filamentation, high-osmolarity responses, cell wall remodeling, and sporulation. (B) Mammalian MAPK modules regulate cell growth, differentiation, stress responses, ... Individual MAPK modules generally can signal independently from each other, and this specificity is manifested in distinct physiologic responses. This is most obvious when studying MAPK signaling in S. cerevisiae. Here a particular extracellular event characteristically activates a specific MAPK module and initiates a unique cellular program (reviewed in references 56 and 77). For example, stimulation of cells with pheromone leads to the activation of the pheromone response pathway (STE11, STE7, and FUS3) (Fig. (Fig.2),2), which ultimately results in cell cycle arrest and the induction of mating-specific genes. However, related MAPKs whose modules share some components with the pheromone response pathway are not affected by pheromone stimulation but are activated only in response to the appropriate stimulus. For example, under conditions of high osmolarity Ste11 can lead to activation of Hog1 but does not induce mating-specific genes. Conversely, conditions that activate the filamentation pathway (which utilizes STE11 and STE7) induce only genes that regulate filamentous growth without triggering pheromone responses or responses to high osmolarity. These observations suggest that yeast cells have developed efficient mechanisms to generate pathway specificity and to successfully suppress cross talk, even when individual components participate in more than one signaling pathway. FIG. 2 Scaffold and adapter molecules in MAPK pathways. MAPK scaffolds and adapters (gray shading) are thought to promote the formation of oligomeric protein complexes with components that function in a specific MAPK module. Scaffolds have been identified in ... In metazoan cells the problem is more complex because each cell is simultaneously exposed to multiple extracellular signals and must integrate these inputs to choose an appropriate response. Thus, the biological context of a signal plays a determinative role in the way that MAPK activation is interpreted. For example, although ERKs generally regulate cell growth and cell differentiation and JNKs participate in a stress response, this is not always the case and in certain cell types activation of JNKs can induce proliferation (110). This indicates that in mammalian systems physiologic responses associated with a certain MAPK module can be cell type specific. Moreover, in PC12 cells, transient stimulation of the ERK cascade leads to proliferation whereas sustained stimulation leads to differentiation, as measured by neurite outgrowth (81). Thus, activation of the ERK cascade can lead to contrasting physiological responses in the same cellular context, suggesting that signal specificity is also determined by regulatory mechanisms other than the selective activation of a MAPK module. In this short review, we outline recent advances in understanding of this signaling system that help to explain how MAPK cascades are regulated and how specificity can be generated. Because of the power of yeast genetics, understanding of MAPK signaling in S. cerevisiae is at an advanced level, and thus many examples that utilize this organism are given. However, analogous mechanisms appear to be operative in metazoans as well. We discuss in turn the role of enzyme-substrate interactions, scaffolding proteins, subcellular targeting and localization, temporal regulation, and signal integration in determining the biological outcome of MAPK activation.

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Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

1996-12-01

Philip James , John T. Halladay , Elizabeth A. Craig

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has … The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

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Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

1998-12-04

Mark S. Longtine , Amos Mckenzie , Douglas J. DeMarini +5 more

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows … An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.

A novel genetic system to detect protein–protein interactions

1989-07-01

Stanley Fields , Ok-kyu Song

Heterologous modules for efficient and versatile PCR-based gene targeting inSchizosaccharomyces pombe

1998-07-01

Jürg Bähler , Jian‐Qiu Wu , Mark S. Longtine +6 more

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the … We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.

Three new dominant drug resistance cassettes for gene disruption inSaccharomyces cerevisiae

1999-10-01

Alan L. Goldstein , John H. McCusker

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, … Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kanr) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6–kanMX3 and pFA6–kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright © 1999 John Wiley & Sons, Ltd.

The genome sequence of the rice blast fungus Magnaporthe grisea

2005-04-01

Ralph A. Dean , Nicholas J. Talbot , Daniel J. Ebbole +7 more

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft … Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation. The genome sequence of the most destructive pathogen of rice is now available. The rice blast fungus Magnaporthe grisea is the first fungal plant pathogen genome to be characterized, and with the rice genome already sequenced, it provides a unique opportunity to study the relationship between host and pathogen. Early findings include a family of novel G-protein-coupled receptors involved in disrupting host defences, a candidate target for fungicides specific for this pest. The genome has been invaded by other genetic elements in the past, probably contributing to rapid evolution when faced with newly introduced resistant rice varieties.

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Guide to Yeast Genetics and Molecular Biology

1993-07-01

C. P. Kurtzman , Christine Guthrie , Gerald R. Fink

This volume and its companion, Volume 350, are specifically designed to meet the needs of graduate students and postdoctoral students as well as researchers, by providing all the up-to-date methods … This volume and its companion, Volume 350, are specifically designed to meet the needs of graduate students and postdoctoral students as well as researchers, by providing all the up-to-date methods necessary to study genes in yeast. Procedures are included that enable newcomers to set up a yeast laboratory and to master basic manipulations. Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines. Specific topics addressed in this book include cytology, biochemistry, cell fractionation, and cell biology.

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

2002-01-01

Yuen Ho , Albrecht Gruhler , Adrian Heilbut +7 more

Proteome survey reveals modularity of the yeast cell machinery

2006-01-22

Anne‐Claude Gavin , Patrick Aloy , Paola Grandi +7 more

Sequencing and comparison of yeast species to identify genes and regulatory elements

2003-05-01

Manolis Kellis , Nick Patterson , Matthew G. Endrizzi +2 more

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

1980-08-01

Peter Novick , C Field , Randy Schekman

A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae

2000-02-01

Peter Uetz , Loïc Giot , Gerard Cagney +7 more

Signal Transduction by the JNK Group of MAP Kinases

2000-10-01

Roger J. Davis

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Improved method for high efficiency transformation of intact yeast cells

1992-01-01

Daniel Gietz , Andrew St. Jean , Robin A. Woods +1 more

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Multifunctional yeast high-copy-number shuttle vectors

1992-01-01

Thomas Christianson , Robert Sikorski , Michael Dante +2 more

Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

1995-04-01

Dominik Mumberg , Rolf Müller , Martin Funk

Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human

1999-01-01

Christian Widmann , Spencer B. Gibson , Matthew Jarpe +1 more

Widmann, Christian, Spencer Gibson, Matthew B. Jarpe, and Gary L. Johnson. Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human. Physiol. Rev. 79: 143–180, 1999. — Mitogen-activated … Widmann, Christian, Spencer Gibson, Matthew B. Jarpe, and Gary L. Johnson. Mitogen-Activated Protein Kinase: Conservation of a Three-Kinase Module From Yeast to Human. Physiol. Rev. 79: 143–180, 1999. — Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. Mitogen-activated protein kinases are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK. Currently, there have been 14 MKKK, 7 MKK, and 12 MAPK identified in mammalian cells. The mammalian MAPK can be subdivided into five families: MAPK erk1/2 , MAPK p38 , MAPK jnk , MAPK erk3/4 , and MAPK erk5 . Each MAPK family has distinct biological functions. In Saccharomyces cerevisiae, there are five MAPK pathways involved in mating, cell wall remodelling, nutrient deprivation, and responses to stress stimuli such as osmolarity changes. Component members of the yeast pathways have conserved counterparts in mammalian cells. The number of different MKKK in MAPK modules allows for the diversity of inputs capable of activating MAPK pathways. In this review, we define all known MAPK module kinases from yeast to humans, what is known about their regulation, defined MAPK substrates, and the function of MAPK in cell physiology.

New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae

1994-12-01

Achim Wach , Arndt Brachat , Rainer Pöhlmann +1 more

Abstract We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open … Abstract We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading‐frame of the E. coli transposon Tn 903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading‐frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3′ end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in‐frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10–3–10–4. The 1·4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR‐added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin‐resistant colonies carried the correctly integrated kanMX module.

The genome sequence of Schizosaccharomyces pombe

2002-02-21

Valerie Wood , Rhian Gwilliam , M.-A. Rajandream +7 more

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Heterologous protein expression in the methylotrophic yeastPichia pastoris

2000-01-01

Joan Lin Cereghino , James M. Cregg

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of … During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

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Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants

2001-12-14

Amy H.Y. Tong , Marie Evangelista , Ainslie B. Parsons +7 more

In Saccharomyces cerevisiae , more than 80% of the ∼6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, … In Saccharomyces cerevisiae , more than 80% of the ∼6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of ∼4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization ( BNI1 , ARP2 , ARC40 , BIM1 ), DNA synthesis and repair ( SGS1 , RAD27 ), or uncharacterized functions ( BBC1 , NBP2 ) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.

All ras proteins are polyisoprenylated but only some are palmitoylated

1989-06-01

John F. Hancock , Anthony I. Magee , Julie E. Childs +1 more

The Transcriptional Program of Sporulation in Budding Yeast

1998-10-23

Shelley Chu , Joseph L. DeRisi , Michael B. Eisen +4 more

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used … Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

Genomic Expression Programs in the Response of Yeast Cells to Environmental Changes

2000-12-01

Audrey P. Gasch , Paul T. Spellman , Camilla M. Kao +5 more

We explored genomic expression patterns in the yeastSaccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast … We explored genomic expression patterns in the yeastSaccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (∼ 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

1999-08-06

Elizabeth A. Winzeler , Daniel Shoemaker , Anna Astromoff +7 more

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae … The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Functional profiling of the Saccharomyces cerevisiae genome

2002-07-25

Guri Giaever , Angela Chu , Li Ni +7 more

Designer deletion strains derived fromSaccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications

1998-01-30

Carrie Baker Brachmann , Adrian Davies , Gregory J. Cost +4 more

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed … A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.

The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification

2001-07-01

Óscar Puig , Friederike Caspary , Guillaume Rigaut +5 more

Two TOR Complexes, Only One of which Is Rapamycin Sensitive, Have Distinct Roles in Cell Growth Control

2002-09-01

Robbie Loewith , Estela Jacinto , Stephan Wullschleger +6 more

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Life with 6000 Genes

1996-10-25

A. Goffeau , B. G. Barrell , Howard Bussey +7 more

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal … The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

A versatile toolbox for PCR‐based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

2004-08-01

Carsten Janke , Maria M. Magiera , Nicole Rathfelder +7 more

Abstract Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae . This … Abstract Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae . This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR‐primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C‐terminal tagged genes or for the exchange of the promoter and N‐terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C‐terminal epitope tags as well as nine different promoter substitutions in combination with N‐terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast‐optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N‐ and C‐terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost‐effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches. Copyright © 2004 John Wiley & Sons, Ltd.

New insights on trehalose: a multifunctional molecule

2003-01-22

Alan D. Elbein

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an α,α-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, … Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an α,α-1,1-glycosidic linkage. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon. In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth. In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation. Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures. There are now at least three different pathways described for the biosynthesis of trehalose. The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP. This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ). Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E. coli) that converts the trehalose-P to free trehalose. A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-α1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose. This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product. A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the α1,4-linkage to an α,α1,1-bond. A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule. Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose. This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose. Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells. This enzyme may be important in lowering trehalose concentrations once the stress is alleviated. Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.

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Osmotic Stress Signaling and Osmoadaptation in Yeasts

2002-06-01

Stefan Hohmann

SUMMARY The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is … SUMMARY The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects.

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The genome sequence of the filamentous fungus Neurospora crassa

2003-04-01

James E. Galagan , Sarah E. Calvo , Katherine A. Borkovich +7 more

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately … Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes--more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca2+ signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

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Integrated Genomic and Proteomic Analyses of a Systematically Perturbed Metabolic Network

2001-05-04

Trey Ideker , Vésteinn Thórsson , Jeffrey A. Ranish +7 more

We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, … We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways.

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

1989-05-01

R Sikorski , P. Hieter

Abstract A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to … Abstract A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

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Global analysis of protein localization in budding yeast

2003-10-01

Won‐Ki Huh , James V. Falvo , Luke C. Gerke +4 more

Correlation between Protein and mRNA Abundance in Yeast

1999-03-01

Steven P. Gygi , Yvan Rochon , B. Robert Franza +1 more

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast … We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243-251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

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[56] Molecular genetic analysis of fission yeast Schizosaccharomyces pombe

1991-01-01

Sergio Moreno , Amar J. S. Klar , Paul Nurse

[1] Getting started with yeast

1991-01-01

Fred Sherman

Change of venue: fission-yeast cell-division cues actually initiate in the nucleus

2025-06-25

Julia Kamenz , James E. Ferrell | Nature

Moesziomyces antarcticus MMF1 Has a Role in the Secretion of Mannosylerythritol Lipids

2025-06-24

Yuze Xu , David A. Stuart | Microorganisms

Mannosyl erythritol lipids (MELs) are glycolipid biosurfactants produced by Ustilaginomycete yeasts. The MEL biosynthetic pathway has been characterized in Ustilago maydis where a putative transporter encoded by MMF1 is required … Mannosyl erythritol lipids (MELs) are glycolipid biosurfactants produced by Ustilaginomycete yeasts. The MEL biosynthetic pathway has been characterized in Ustilago maydis where a putative transporter encoded by MMF1 is required for the secretion of the glycolipid surfactant to the extracellular space. The anamorphic yeast Moesziomyces antarcticus is a prolific producer of MELs, but the mechanism of MEL secretion is less well characterized than in U. maydis. Homologous recombination was employed to generate a disruption of the MMF1 gene in M. antarcticus JCM10317. This mutation did not prevent the intracellular accumulation of MEL species but did result in significantly reduced secretion of the conventional MEL-A, MEL-B and MEL-C species detectable by thin-layer chromatography. However, the mutant strain did secrete a glycolipid species that is distinct from conventional MEL-A/B/C and similar to a glycolipid secreted by MMF1 mutant strains of U. maydis and Pseudozyma tsukubaensis. Despite the defect in MEL secretion displayed by the M. antarcticus strain harbouring a disrupted MMF1 gene, these cells did not display a significant defect in growth or cell morphology. The findings of this investigation provide evidence that M. antarcticus MMF1 encodes a transporter required for the secretion of MELs but not required for MEL synthesis or cell growth.

The Cys2-His2 zinc finger transcription factor NsdC regulates hyphal branching, conidiation and mycelial pellet morphology in Aspergillus oryzae

2025-06-23

Hui Ting Chu , Naazneen Sofeo , Jasmin Jebarani Jebamony +1 more | Fungal Genetics and Biology

Decapeptide Inducer Promotes the Conidiation of Phytopathogenic Magnaporthe oryzae via the Mps1 MAPK Signaling Pathway

2025-06-19

Mengya Yang , Yanan Liu , Jianhua Qi | International Journal of Molecular Sciences

Magnaporthe oryzae (M. oryzae) is a phytopathogenic fungus that inflicts damage on vital crops, particularly rice. Its asexual reproduction leads to the generation of numerous conidia, which is a critical … Magnaporthe oryzae (M. oryzae) is a phytopathogenic fungus that inflicts damage on vital crops, particularly rice. Its asexual reproduction leads to the generation of numerous conidia, which is a critical factor contributing to the prevalence of rice blast disease. However, the molecules regulating the asexual reproduction of M. oryzae are unknown. In our study, to identify the molecules capable of regulating the asexual reproduction of M. oryzae, compositions of the complete medium (CM) were screened. Results showed that acid-hydrolyzed casein (AHC) could remarkably promote conidial production. One M. oryzae conidiation inducer was isolated from AHC using high-performance liquid chromatography (HPLC) under the guidance of bioassay. Its structure was further elucidated as a decapeptide compound (pyroGlu-EQNQEQPIR) by LC-MS/MS, chemical synthesis, and conidium-inducing assays, named M. oryzae conidiation inducer decapeptide (MCIDP). MCIDP could significantly promote the conidiation of M. oryzae and two other filamentous ascomycetes (Botrytis cinerea and Fusarium graminearum). The Mps1 MAPK cascade signaling pathway is crucial for conidiation, and the effect of MCIDP on this pathway was investigated to elucidate the mechanism underlying conidiation enhancement. qRT-PCR analysis demonstrated that MCIDP could remarkably upregulate the gene expression within the Mps1 MAPK cascade signaling pathway, especially the WSC2, WSC3, PKC1, MKK1, MPS1, and MIG1. Furthermore, the ΔMowsc1, ΔMowsc2, ΔMowsc3, and ΔMomid2 mutant strains were constructed. Bioassay results showed that MCIDP failed to promote conidial formation and hyphal growth in these mutant strains. These findings indicate that MCIDP promotes conidiation of M. oryzae by modulating the Mps1 MAPK signaling pathway.

Correction: A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway

2025-06-18

Gabriella Gerlach , Rachel Carrock , Robyn Stix +2 more | PLoS Computational Biology

[This corrects the article DOI: 10.1371/journal.pcbi.1007815.]. [This corrects the article DOI: 10.1371/journal.pcbi.1007815.].

Divergence in a eukaryotic transcription factor’s co-TF dependence involves multiple intrinsically disordered regions

2025-06-18

Lindsey F. Snyder , Emily M O'Brien , Jiajun Zhao +7 more | Nature Communications

Beyond division and morphogenesis: Considering the emerging roles of septins in plasma membrane homeostasis and cell wall integrity in human fungal pathogens

2025-06-17

Stephani Martinez Barrera , Lukasz Kozubowski | PLoS Pathogens

Basal association of a transcription factor favors early gene expression

2025-06-16

Sandrine Pinheiro , Mariona Nadal‐Ribelles , Carme Solé +4 more | PLoS Genetics

Responses to extracellular signals via Mitogen-Activated Protein Kinase (MAPK) pathways control complex transcriptional programs where hundreds of genes are induced at a desired level with a specific timing. Gene expression … Responses to extracellular signals via Mitogen-Activated Protein Kinase (MAPK) pathways control complex transcriptional programs where hundreds of genes are induced at a desired level with a specific timing. Gene expression regulation is largely encoded in the promoter of the gene, which harbors numerous transcription factor binding sites. In the mating MAPK pathway of Saccharomyces cerevisiae , one major transcription factor, Ste12, controls the chronology of gene expression necessary for the fusion of two haploid cells. Because endogenous promoters encode a large diversity of Ste12 binding sites (PRE), we engineered synthetic promoters to decipher the rules that dictate mating gene induction. Conformations of PRE dimers that allow efficient gene expression were identified. The strength of binding of Ste12 to the PRE and the distance of the binding sites to the core promoter modulate the level of induction. The speed of activation is ensured by favoring a basal association of Ste12 by using a strong dimer of PRE located in a nucleosome depleted region.

The Obg-Like ATPase Ola1 prevents excessive mitochondrial reactive oxygen species by inhibiting MAPK/Pmk1 signaling in fission yeast

2025-06-16

Shuping Luo , Mengdan Zhu , Chuanhai Fu | Microbiological Research

The Aspergillus nidulans velvet domain containing transcription factor VeA is shuttled from cytoplasm into nucleus during vegetative growth and stays there for sexual development, but has to return into cytoplasm for asexual development

2025-06-16

Anja Strohdiek , Anna M. Köhler , Rebekka Harting +7 more | PLoS Genetics

Survival of multicellular organisms requires the coordinated interplay between networks regulating gene expression and controlled intracellular transport of respective regulators. Velvet domain proteins are fungal transcription factors, which form various … Survival of multicellular organisms requires the coordinated interplay between networks regulating gene expression and controlled intracellular transport of respective regulators. Velvet domain proteins are fungal transcription factors, which form various heterodimers and play key roles in controlling early developmental decisions towards more either asexual or sexual differentiation. VeA is the central subunit of the trimeric velvet complex VelB-VeA-LaeA, which links transcriptional to epigenetic control for the coordination of fungal developmental programs to specific secondary metabolite synthesis. Nuclear localization of the VeA bridging factor is carefully controlled in fungi. In this work we demonstrate that VeA carries three nuclear localization signals NLS1, NLS2 and NLS3, which all contribute to nuclear import. We show that VeA has an additional nuclear export sequence (NES) which provides a shuttle function to allow the cell to relocate VeA to the cytoplasm. VeA is nuclear during vegetative growth, but has to be exported from the nucleus to allow and promote asexual development. In contrast, progression of the sexual pathway requires continuous nuclear VeA localization. Our work shows that an accurate nuclear import and export control of velvet proteins is further connected to specific stability control mechanism as prerequisites for fungal development and secondary metabolism. These results illustrate the various complex mutual dependencies of velvet regulatory proteins for coordinating fungal development and secondary metabolism.

N-Terminus of Cid14 Activates RNA Unwinding by Mtr4 in the Schizosaccharomyces pombe TRAMP Complex

2025-06-16

Mark D. Gold , Matthew D. Staten , Kevin Morgan +2 more | Biochemistry

The TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex is a multiprotein assembly that targets RNA substrates for processing or degradation by the nuclear exosome. In Saccharomyces cerevisiae, the helicase activity of Mtr4 is … The TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex is a multiprotein assembly that targets RNA substrates for processing or degradation by the nuclear exosome. In Saccharomyces cerevisiae, the helicase activity of Mtr4 is enhanced by TRAMP assembly. Here we identify unexpected species-specific differences in the ability of Mtr4 to unwind a model RNA substrate. Unlike S. cerevisiae Mtr4, Schizosaccharomyces pombe Mtr4 retains RNA-stimulated ATPase activity, but is unable to unwind a model RNA substrate. This decoupling of ATPase and helicase activity is overcome by TRAMP formation. We further demonstrate that activation of helicase activity is accomplished by unique interactions with multiple regions of the intrinsically disordered N-terminus of the poly(A) polymerase, Cid14 (the S. pombe homologue of S. cerevisiae Trf4). Finally, we propose a model where Mtr4 adaptor complexes regulate unwinding activity by coordinating interdomain interactions within the helicase core.

Phase separation of a master transcription factor targeted by a natural compound regulates fungal ergosterol homeostasis

2025-06-12

Huiyuan Wang , Yuhua Wang , Jingrui Wang +6 more | Science China Life Sciences

Structural and regulatory dynamics of septum development in fungal hyphae

2025-06-03

Olga A. Callejas‐Negrete , Alejandro Fajardo-Peralta , Rosa R. Mouriño‐Pérez | Fungal Genetics and Biology

CBC Complex Regulates Hyphal Growth, Sclerotial Quantity, and Pathogenicity in the Necrotrophic Fungus Botrytis cinerea

2025-06-02

Yinshan Zhang , Xueting Chen , Guihua Li +3 more | Journal of Fungi

The cap-binding protein complex (CBC), comprising Cbp20 and Cbp80, is crucial for gene expression, yet its role in the notorious crop pathogen Botrytis cinerea remains unclear. Immunoprecipitation coupled with LC-MS/MS … The cap-binding protein complex (CBC), comprising Cbp20 and Cbp80, is crucial for gene expression, yet its role in the notorious crop pathogen Botrytis cinerea remains unclear. Immunoprecipitation coupled with LC-MS/MS demonstrated that BcCbp20 interacts with BcCbp80. Yeast two-hybrid, GST pull-down, and Split-luciferase complementation assays confirmed that the conserved RNA recognition motif (RRM, 54–127 aa) of BcCbp20 and the N-terminal MIF4G domain (1–370 aa, 1–577 aa) of BcCbp80 constitute the core interaction regions. Genetic transformation experiments revealed that BcCBP80 exerts a more dominant role than BcCBP20 in regulating hyphal morphology, growth rate, conidiophore development, and conidial yield. Furthermore, BcCBP20 and BcCBP80 differentially regulate sclerotium formation to maintain sclerotial quantity. Based on pathogenicity assays, BcCBP80 associated with infection cushion development, with this phenotypic alteration possibly being among the factors correlated with altered pathogenicity. However, the increased sensitivity of ΔBccbp20 to various stress factors may be the primary reason for the diminished pathogenicity. Taken together, these results indicate that BcCBP20 and BcCBP80 play important roles in multiple aspects of B. cinerea growth, development, stress response, and pathogenicity.

Multi-omics Analysis of the Active Components and Mechanisms of Baojin Chenfei Formula in Silica-Induced Murine Silicosis

2025-06-01

He WR , Lu Yq , Liu XG +5 more | Phytomedicine

The roles of the cytoskeleton in fungal tip growth: Insights from aspergillus nidulans

2025-06-01

Berl R. Oakley , Miguel Á. Peñalva | Fungal Genetics and Biology

Roles and transcriptional regulation of the endogenous cellulases in association with substrate and cellular processes in a thermoacidophilic archaeon

2025-06-01

Shuai Li , Qihong Huang , Yunfeng Yang +4 more | Bioresource Technology

The B-type cyclin Clb4 prevents meiosis I sister centromere separation in budding yeast

2025-05-30

Gal Lumbroso , Gisela Cairo , Soni Lacefield +1 more | G3 Genes Genomes Genetics

Abstract In meiosis, one round of DNA replication followed by two rounds of chromosome segregation halves the ploidy of the original cell. Accurate chromosome segregation in meiosis I depends on … Abstract In meiosis, one round of DNA replication followed by two rounds of chromosome segregation halves the ploidy of the original cell. Accurate chromosome segregation in meiosis I depends on recombination between homologous chromosomes. Sister centromeres attach to the same spindle pole in this division and only segregate in meiosis II. We used budding yeast to select for mutations that produced viable spores in the absence of recombination. The most frequent mutations inactivated CLB4, which encodes one of four B-type cyclins. In two wild yeast isolates, Y55 and SK1, but not the W303 laboratory strain, deleting CLB4 causes premature sister centromere separation and segregation in meiosis I and frequent termination of meiosis after a single division, demonstrating a novel role for Clb4 in meiotic chromosome dynamics and meiotic progression. This role depends on the genetic background since meiosis in W303 is largely independent of CLB4.

Septin higher-order structure on yeast membranes in vitro

2025-05-30

James A. Goodchild , Brandy N. Curtis , Yangang Pan +4 more | Nature Communications

Promoting cell fitness and longevity in fission yeast – The role of nutrient deprivation pathways on mitochondrial efficiency

2025-05-30

Montserrat Rojo de la Vega , Ferran Gómez-Armengol , José Ayté +1 more | Free Radical Biology and Medicine

The bur1-107 mutant delays G1/S transition and alleviates hydroxyurea sensitivity in checkpoint-deficient yeast

2025-05-30

Stefany Cristine Rodrigues da Silva , Francisco Meirelles Bastos de Oliveira | Genetics

Abstract In Saccharomyces cerevisiae, the cyclin-dependent kinase Bur1 is primarily known for its role in promoting transcription elongation, thereby regulating gene expression. In this study, we investigated the genetic interactions … Abstract In Saccharomyces cerevisiae, the cyclin-dependent kinase Bur1 is primarily known for its role in promoting transcription elongation, thereby regulating gene expression. In this study, we investigated the genetic interactions between a hypomorphic BUR1 allele (bur1-107) and null mutants of the checkpoint kinases Mec1 and Rad53. Remarkably, bur1-107 alleviated the sensitivity of mec1 and rad53 mutants to hydroxyurea (HU), suggesting that Bur1 activity becomes detrimental when checkpoint signaling is impaired. Furthermore, the bur1-107 mutation delayed the G1-to-S phase transition, implicating Bur1 as a key player in cell cycle progression. In HU-treated mec1 mutants, bur1-107 reduced γ-H2A accumulation, promoted S-phase resumption, and suppressed the formation of sub-G1 populations. Together, these findings suggest that Bur1-driven G1-to-S phase progression exacerbates DNA damage and cell death in checkpoint-deficient cells exposed to HU. This study highlights a novel role for Bur1 in modulating the cellular response to replication stress in checkpoint-compromised cells.

Varied mutual growth inhibition between commensal yeasts and <E. coli> strains

2025-05-29

Eugénie Pingault--Ordronneau , A François , Vashineta Ragoobeer +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Interkingdom interactions between bacteria and fungi are an emerging research field that provides insights into pathological, environmental, and microbiota-related relationships. However, the mechanisms governing these interactions, particularly in the context … Interkingdom interactions between bacteria and fungi are an emerging research field that provides insights into pathological, environmental, and microbiota-related relationships. However, the mechanisms governing these interactions, particularly in the context of microbial resistance, remain largely unknown. This study aims to enhance our understanding of the complex interactions between different Candida , Nakaseomyces and Sacharomyces species from the human microbiota and two not isogenic strains of Escherichia coli (antibiotic-susceptible E. coli -ATCC and multidrug-resistant E. coli -OXA48). Forty-nine Candida strains were co-cultured with the two E. coli strains. Both bacterial and yeast growth was monitored using flow cytometry and compared to monocultures. The effect of yeast culture supernatants on E. coli proliferation was also investigated. Metabolomic fingerprints and metabolite identification were performed using mass spectrometry-based approaches followed by multiblock statistical analyses. The inhibitory powers (IP) of yeasts against E. coli and vice versa varied significantly among fungal species. N. glabrata exhibited the strongest inhibition against E. coli -ATCC, while Candida lusitaniae , C. kefyr , C. krusei , C. tropicalis , and C. dubliniensis showed lower IPs. C. parapsilosis and Saccharomyces cerevisiae had no inhibitory effects. Against E. coli -OXA48, most yeasts displayed no inhibition, except for N. glabrata . Conversely, E. coli inhibited yeast growth more effectively, particularly Candida albicans . Fungal supernatants from S. cerevisiae , C. lusitaniae , and N. glabrata showed the highest inhibitory effects on E. coli -ATCC, while S. cerevisiae , C. krusei , and C. lusitaniae were most effective against E. coli -OXA48. Unsupervised metabolite profiling data analysis with multiblock approach highlighted a clustering of samples according to yeast species. Regarding inhibitory power on E. coli (ATCC or OXA48), active supernatants tend to cluster together suggesting the presence of similar metabolites; some were further characterized. This study highlights the diverse interactions between E. coli and commensal yeasts. From an applied perspective, these findings pave the way for identifying probiotics or postbiotics with potential applications in combating multidrug-resistant bacteria through novel antimicrobial compounds.

Quantitative Analysis of Stress Tolerance in Kveik Yeast

2025-05-29

Daishi Shirasaya , Chris Powell | Journal of the American Society of Brewing Chemists

Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae

2025-05-29

Yingying Cai , Xue‐Ming Zhu , Muhammad Noman +7 more | Cell Communication and Signaling

Autophagy is crucial for appressorium development and host invasion by phytopathogenic fungi, including Magnaporthe oryzae. During appressorium maturation, many organelles, such as nuclei, in the conidia need to be degraded … Autophagy is crucial for appressorium development and host invasion by phytopathogenic fungi, including Magnaporthe oryzae. During appressorium maturation, many organelles, such as nuclei, in the conidia need to be degraded through autophagy to be recycled in appressorium. However, the interplay between autophagy and nuclear membrane systems remains poorly understood. In this study, we functionally characterized MoNup50, a nuclear pore-associated protein. Despite sharing limited sequence identity with human and yeast Nup proteins, MoNup50 contains conserved domains typical of nuclear pore complex proteins. Observation under fluorescence microscopy revealed that MoNup50 localizes at the nuclear membrane in M. oryzae. Deletion of MoNUP50 resulted in reduced hyphal growth, spore production, appressorium formation, and pathogenicity, while increasing sensitivity to osmotic stress and cell wall disruption. Notably, MoNup50 interacts with the key autophagy protein MoAtg7, which regulates MoAtg8-PE synthesis during autophagy. Moreover, MoNUP50 deletion led to elevated autophagy levels and increased phosphorylation of the MAPKs Osm1 and Mps1. These findings suggest that MoNup50 is involved in appressorium morphogenesis and pathogenicity by modulating autophagy and MAPK pathways, highlighting the critical role of nuclear pore proteins in M. oryzae pathogenicity and their potential cross-talk with autophagic and MAPK signaling.

Woronin body protein UvHex1 is essential for pathogenesis, and contributes to conidiation, cell stress response, lipid metabolism, and mycotoxins production in rice false smut fungus Ustilaginoidea virens

2025-05-29

Yijun Mao , Xiayan Pan , Mina Yu +7 more | Phytopathology Research

Abstract Woronin bodies (WBs) are unique single-membrane microbodies derived from peroxisomes found in fungi within the Pezizomycotina subphylum. WBs can seal hyphal septa to prevent cytoplasmic leakage upon hyphal damage. … Abstract Woronin bodies (WBs) are unique single-membrane microbodies derived from peroxisomes found in fungi within the Pezizomycotina subphylum. WBs can seal hyphal septa to prevent cytoplasmic leakage upon hyphal damage. The hexagonal peroxisomal protein, Hex1, is a key component in the formation of WBs. In this study, the ortholog of Hex1 in U. virens (UvHex1) was targeted for deletion using homologous recombination and subsequent complementation. The findings revealed that the deletion of Uvhex1 gene resulted in a loss of pathogenicity, a slight reduction in mycelial growth, and highly sensitivity to cell wall, oxidative, and osmotic stress. In contrast, the Uvhex1 deletion mutants exhibited enhanced capacity for utilizing long-chain fatty acids and producing mycotoxins. These results suggest that UvHex1 is crucial for WBs formation and pathogenicity, and it also plays important roles in conidiation, cellular stress response, lipid metabolism, and mycotoxin production in the rice false smut fungus U. virens .

Mechanisms and Strategies for Engineering Oxidative Stress Resistance in Saccharomyces cerevisiae

2025-05-28

Taotao Feng , Hongwei Yu , Lidan Ye | Chem & Bio Engineering

Molecular Evolution of the members of the Snq2/Pdr18 subfamily of Pdr transporters in the Hemiascomycete yeasts

2025-05-27

Paulo Jorge Dias

Abstract The transporters of the ATP-Binding Cassette (ABC) Superfamily involved in the Multidrug Resistance (MDR) phenomena are also known as ABC Pleiotropic Drug Resistance (PDR) proteins. The homologs of the … Abstract The transporters of the ATP-Binding Cassette (ABC) Superfamily involved in the Multidrug Resistance (MDR) phenomena are also known as ABC Pleiotropic Drug Resistance (PDR) proteins. The homologs of the Saccharomyces cerevisiae SNQ2 and PDR18 genes were identified in 171 yeast genomes, representing 68 different hemiascomycetous species. All early-divergent yeast species analyzed in this work lack Snq2/Pdr18 homologs, suggesting that the origin of these ABC-PDR genes in hemiascomycete yeasts resulted from a horizontal transfer event. The evolutionary pathway of the Snq2/Pdr18 protein subfamily in pathogenic Candida species was also reconstructed, revealing a main gene lineage leading to the C. albicans SNQ2 gene. The results indicate that, after the gene duplication event at the origin of the SNQ2/PDR18 paralogs, the PDR18 ortholog has been under strong diversifying selection and suggest that a small portion of the sequence of the SNQ2 ancestral ortholog might have been under mild positive selection. The results also showed that strong positive selection was exerted over one of the two paralogs generated by the Whole Genome Duplication (WGD) event, corresponding to the duplicate at the origin of a “short-lived” WGD sublineage.

VdAHA1 positively regulate pathogenicity in Verticillium dahliae

2025-05-26

Dongpo Li , Yuan Yuan , Jinglong Zhou +6 more

AHA1 (activator of HSP90 ATPase) is a co-chaperone protein that mainly performs its function by interacting with the HSP90. The biological function of AHA1 has been widely reported in many … AHA1 (activator of HSP90 ATPase) is a co-chaperone protein that mainly performs its function by interacting with the HSP90. The biological function of AHA1 has been widely reported in many species. In this study, we knocked out the VdAHA1 gene of V. dahliae by homologous recombination method. The VdAHA1 knockout mutants showed increased drug sensitivity to ergosterol synthesis pathway, significantly inhibiting ergosterol biosynthesis. The VdAHA1 knockout mutant strain also showed decreased melanin in microsclerotia by reduced expression of microsclerotia and melanin related genes Vaflm, Vayg1 , and VdSCD . The VdAHA1 mutant showed decreased conidial production that were slightly damaged and showed more sensitivity to abiotic stresses such as temperature, SDS, CR, Sorbitol (SBT), NaCl, and KCl and decreased ATP contents. More importantly, the mutant was significantly less virulent to cotton than the wild type. This study identified the important functions of VdAHA1 in the growth, stress resistance, and virulence.

Stability and functional consequences of disulfide bond engineering in Aspergillus flavus uricase

2025-05-26

Mohammad Reza Rahbar , Navid Nezafat , Mohammad Hossein Morowvat +4 more

Disulfide bond engineering is a promising strategy for enhancing the stability and functional lifespan of enzymes in therapeutic and industrial applications. In this study, we applied computational modeling to introduce … Disulfide bond engineering is a promising strategy for enhancing the stability and functional lifespan of enzymes in therapeutic and industrial applications. In this study, we applied computational modeling to introduce interchain disulfide bonds in Aspergillus flavus uricase to increase its stability without compromising catalytic efficiency. Six uricase muteins were engineered with targeted disulfide bonds at positions selected based on energetic frustration, structural integrity, and tunnel profiling analyses. By employing frustration density mapping, Root Mean Square Fluctuation (RMSF) profiling, and tunnel analysis, we evaluated the structural stability, flexibility, and substrate accessibility of each variant. Our findings revealed that muteins with disulfide bonds between residues such as Ala6-Cys290 and Ser119-Cys220 exhibited significant reductions in highly frustrated regions, enhancing the enzyme's structural resilience. RMSF analysis indicated decreased local flexibility near disulfide sites, contributing to increased stability. Tunnel profiling further demonstrated that muteins with strategically placed disulfide bonds maintained favorable substrate access and low-energy barriers, critical for catalytic turnover. These results underscore the potential of targeted disulfide bond engineering for optimizing enzyme stability, offering valuable insights for the development of stable, high-performance biocatalysts suitable for therapeutic and industrial use.