scholarly journals Bidirectional-Genetics Platform, a Dual-Purpose Mutagenesis Strategy for Filamentous Fungi

2013 ◽  
Vol 12 (11) ◽  
pp. 1547-1553 ◽  
Author(s):  
Jaejin Park ◽  
Yong-Hwan Lee
Keyword(s):  
Filamentous Fungi ◽  
Vegetative Growth ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Gene Replacement ◽  
Forward Genetics ◽  
Fungal Genome ◽  
Reverse Genetic ◽  
Content Type ◽  
Insertional Mutants

ABSTRACTRapidly increasing fungal genome sequences call for efficient ways of generating mutants to translate quickly gene sequences into their functions. A reverse genetic strategy via targeted gene replacement (TGR) has been inefficient for many filamentous fungi due to dominant production of undesirable ectopic transformants. Although large-scale random insertional mutagenesis via transformation (i.e., forward genetics) facilitates high-throughput uncovering of novel genes of interest, generating a huge number of transformants, which is necessary to ensure the likelihood of mutagenizing most genes, is time-consuming. We propose a new strategy, entitled theBidirectional-Genetics (BiG) platform, which combines both forward and reverse genetic strategies by recycling ectopic transformants derived from TGR as a source for random insertional mutants. The BiG platform was evaluated using the rice blast fungusMagnaporthe oryzaeas a model. Over 10% of >1,000M. oryzaeectopic transformants, generated during disruption of specific genes, displayed abnormality in vegetative growth, pigmentation, and/or asexual reproduction. In this pool of putative mutants, we isolated insertional mutants with mutations in three genes involved in histidine biosynthesis (MoHIS5), vegetative growth (MoVPS74), or conidiophore formation (MoFRQ) (where “Mo” indicates “M. oryzae”), supporting the utility of this platform for systematic gene function studies.

scholarly journals Methylenetetrahydrofolate Reductase Activity Is Involved in the Plasma Membrane Redox System Required for Pigment Biosynthesis in Filamentous Fungi

2010 ◽  
Vol 9 (8) ◽  
pp. 1225-1235 ◽  
Author(s):  
Rasmus J. N. Frandsen ◽  
Klaus Selk Albertsen ◽  
Peter Stougaard ◽  
Jens L. Sørensen ◽  
Kristian F. Nielsen ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Reduction Potential ◽  
Methylenetetrahydrofolate Reductase ◽  
Reductase Activity ◽  
Gene Replacement ◽  
Sequence Motif ◽  
Prolonged Incubation ◽  
Content Type ◽  
Conserved Sequence ◽  
Pigment Biosynthesis

ABSTRACT Methylenetetrahydrofolate reductases (MTHFRs) play a key role in biosynthesis of methionine and S-adenosyl-l-methionine (SAM) via the recharging methionine biosynthetic pathway. Analysis of 32 complete fungal genomes showed that fungi were unique among eukaryotes by having two MTHFRs, MET12 and MET13. The MET12 type contained an additional conserved sequence motif compared to the sequences of MET13 and MTHFRs from other eukaryotes and bacteria. Targeted gene replacement of either of the two MTHFR encoding genes in Fusarium graminearum showed that they were essential for survival but could be rescued by exogenous methionine. The F. graminearum strain with a mutation of MET12 (FgΔMET12) displayed a delay in the production of the mycelium pigment aurofusarin and instead accumulated nor-rubrofusarin and rubrofusarin. High methionine concentrations or prolonged incubation eventually led to production of aurofusarin in the MET12 mutant. This suggested that the chemotype was caused by a lack of SAM units for the methylation of nor-rubrofusarin to yield rubrofusarin, thereby imposing a rate-limiting step in aurofusarin biosynthesis. The FgΔMET13 mutant, however, remained aurofusarin deficient at all tested methionine concentrations and instead accumulated nor-rubrofusarin and rubrofusarin. Analysis of MET13 mutants in F. graminearum and Aspergillus nidulans showed that both lacked extracellular reduction potential and were unable to complete mycelium pigment biosynthesis. These results are the first to show that MET13, in addition to its function in methionine biosynthesis, is required for the generation of the extracellular reduction potential necessary for pigment production in filamentous fungi.

scholarly journals The Aspergillus nidulansnucAEndoGHomologue Is Not Involved in Cell Death

2010 ◽  
Vol 10 (2) ◽  
pp. 276-283 ◽  
Author(s):  
Bárbara de Castro Pimentel Figueiredo ◽  
Patrícia Alves de Castro ◽  
Taísa Magnani Dinamarco ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Cell Death ◽  
Aspergillus Nidulans ◽  
Dna Fragmentation ◽  
Filamentous Fungi ◽  
Large Scale ◽  
Apoptosis Induction ◽  
Content Type ◽  
Endonuclease G ◽  
First Time

ABSTRACTUpon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if theAspergillus nidulanshomologue of the EndoG gene, namednucAEndoG, is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response inA. nidulans.

Transgenesis of schistosomes: approaches employing mobile genetic elements

Parasitology ◽  
2007 ◽  
Vol 135 (2) ◽  
pp. 141-153 ◽  
Author(s):  
V. H. MANN ◽  
M. E. MORALES ◽  
K. J. KINES ◽  
P. J. BRINDLEY
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Draft Genome ◽  
Mobile Genetic Elements ◽  
Forward Genetics ◽  
Loss Of Function ◽  
Genetic Elements ◽  
The Past ◽  
Foreign Genes

SUMMARYDraft genome sequences forSchistosoma mansoniandSchistosoma japonicumare now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

scholarly journals A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
McKenzie Carter ◽  
Stephanie Gomez ◽  
Sam Gritz ◽  
Stephen Larson ◽  
Eugenia Silva-Herzog ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Large Scale ◽  
Resistance Mechanisms ◽  
Genetic Screen ◽  
Forward Genetics ◽  
Gain Of Function ◽  
Content Type ◽  
Genetic Tools ◽  
Gene Functions

ABSTRACT Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library’s usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research. IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

Financial distress determinants among SMEs: empirical evidence from Sweden

2020 ◽  
Vol 47 (3) ◽  
pp. 547-560 ◽  
Author(s):  
Darush Yazdanfar ◽  
Peter Öhman
Keyword(s):  
Financial Crisis ◽  
Financial Distress ◽  
Large Scale ◽  
Global Financial Crisis ◽  
Binary Logistic Regression ◽  
Data Availability ◽  
Cross Sectional ◽  
Data Set ◽  
Content Type ◽  
The Global Financial Crisis

PurposeThe purpose of this study is to empirically investigate determinants of financial distress among small and medium-sized enterprises (SMEs) during the global financial crisis and post-crisis periods.Design/methodology/approachSeveral statistical methods, including multiple binary logistic regression, were used to analyse a longitudinal cross-sectional panel data set of 3,865 Swedish SMEs operating in five industries over the 2008–2015 period.FindingsThe results suggest that financial distress is influenced by macroeconomic conditions (i.e. the global financial crisis) and, in particular, by various firm-specific characteristics (i.e. performance, financial leverage and financial distress in previous year). However, firm size and industry affiliation have no significant relationship with financial distress.Research limitationsDue to data availability, this study is limited to a sample of Swedish SMEs in five industries covering eight years. Further research could examine the generalizability of these findings by investigating other firms operating in other industries and other countries.Originality/valueThis study is the first to examine determinants of financial distress among SMEs operating in Sweden using data from a large-scale longitudinal cross-sectional database.

scholarly journals An FYVE-Domain-Containing Protein, PsFP1, Is Involved in Vegetative Growth, Oxidative Stress Response and Virulence of Phytophthora sojae

2021 ◽  
Vol 22 (12) ◽  
pp. 6601
Author(s):  
Jinhui Zhang ◽  
Xiaoran Du ◽  
Xin Zhou ◽  
Duo Jin ◽  
Jianqiang Miao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Trafficking ◽  
Vegetative Growth ◽  
Genome Mining ◽  
Gene Replacement ◽  
Wide Distribution ◽  
Stress Sensitivity ◽  
Phytophthora Sojae ◽  
Knock Out ◽  
Fyve Domain

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

The dialectics of authoring expansive learning: tracing the long tail of a Change Laboratory

2016 ◽  
Vol 28 (4) ◽  
pp. 245-262 ◽  
Author(s):  
Annalisa Sannino ◽  
Yrjö Engeström ◽  
Johanna Lahikainen
Keyword(s):  
Large Scale ◽  
Scale Transformation ◽  
Dialectical Tensions ◽  
Content Type ◽  
Organization Model ◽  
Expansive Learning ◽  
Conceptual Argument ◽  
University Library ◽  
Dialectical Process ◽  
Cultural Historical Activity

Purpose The paper aims to examine organizational authoring understood as a longitudinal, material and dialectical process of transformation efforts. The following questions are asked: To which extent can a Change Laboratory intervention help practitioners author their own learning? Are the authored outcomes of a Change Laboratory intervention futile if a workplace subsequently undergoes large-scale organizational transformations? Does the expansive learning authored in a Change Laboratory intervention survive large-scale organizational transformations, and if so, why does it survive and how? Design/methodology/approach The paper develops a conceptual argument based on cultural–historical activity theory. The conceptual argument is grounded in the examination of a case of eight years of change efforts in a university library, including a Change Laboratory (CL) intervention. Follow-up interview data are used to discuss and illuminate our argument in relation to the three research questions. Findings The idea of knotworking constructed in the CL process became a “germ cell” that generates novel solutions in the library activity. A large-scale transformation from the local organization model developed in the CL process to the organization model of the entire university library was not experienced as a loss. The dialectical tension between the local and global models became a source of movement driven by the emerging expansive object. Practitioners are modeling their own collective future competences, expanding them both in socio-spatial scope and interactive depth. Originality/value The article offers an expanded view of authorship, calling attention to material changes and practical change actions. The dialectical tensions identified serve as heuristic guidelines for future studies and interventions.

Dynamic output feedback control for nonlinear large-scale interconnected systems

2020 ◽  
Vol 39 (4) ◽  
pp. 801-821
Author(s):  
Ezzeddine Touti ◽  
Ali Sghaier Tlili ◽  
Muhannad Almutiry
Keyword(s):  
Decentralized Control ◽  
Output Feedback ◽  
Large Scale ◽  
Optimization Problem ◽  
Lyapunov Theory ◽  
Interconnected Systems ◽  
Dynamic Output Feedback ◽  
Content Type ◽  
Dynamic Output ◽  
Control Scheme

Purpose This paper aims to focus on the design of a decentralized observation and control method for a class of large-scale systems characterized by nonlinear interconnected functions that are assumed to be uncertain but quadratically bounded. Design/methodology/approach Sufficient conditions, under which the designed control scheme can achieve the asymptotic stabilization of the augmented system, are developed within the Lyapunov theory in the framework of linear matrix inequalities (LMIs). Findings The derived LMIs are formulated under the form of an optimization problem whose resolution allows the concurrent computation of the decentralized control and observation gains and the maximization of the nonlinearity coverage tolerated by the system without becoming unstable. The reliable performances of the designed control scheme, compared to a distinguished decentralized guaranteed cost control strategy issued from the literature, are demonstrated by numerical simulations on an extensive application of a three-generator infinite bus power system. Originality/value The developed optimization problem subject to LMI constraints is efficiently solved by a one-step procedure to analyze the asymptotic stability and to synthesize all the control and observation parameters. Therefore, such a procedure enables to cope with the conservatism and suboptimal solutions procreated by optimization problems based on iterative algorithms with multi-step procedures usually used in the problem of dynamic output feedback decentralized control of nonlinear interconnected systems.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

scholarly journals Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of theStreptococcus pyogenesChromosome

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Dragutin J. Savic ◽  
Scott V. Nguyen ◽  
Kimberly McCullor ◽  
W. Michael McShan
Keyword(s):  
Dna Sequences ◽  
Parental Strain ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Acute Infection ◽  
Relative Length ◽  
Published Data ◽  
Rich Medium ◽  
Content Type

ABSTRACTA large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, bothin vitroandin vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place nearoriCalways happen to secure the linkage oforiCto DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of theoriCand the chromosome partition cluster is preserved.

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