scholarly journals High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

2016 ◽  
Vol 90 ◽  
pp. 1-11 ◽  
Author(s):  
Ailisa Blum ◽  
Aurélie H. Benfield ◽  
Jiri Stiller ◽  
Kemal Kazan ◽  
Jacqueline Batley ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Fusarium Graminearum ◽  
High Throughput ◽  
Gain Of Function ◽  
Mutant Screen

scholarly journals A simple high-throughput technology enables gain-of-function screening of human microRNAs

BioTechniques ◽  
2013 ◽  
Vol 54 (2) ◽  
Author(s):  
Wen-Chih Cheng ◽  
Tami J. Kingsbury ◽  
Sarah J. Wheelan ◽  
Curt I. Civin
Keyword(s):  
High Throughput ◽  
Gain Of Function ◽  
High Throughput Technology

scholarly journals Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells

2020 ◽  
Author(s):  
Seyad Arad Moghadasi ◽  
Jordan T. Becker ◽  
Christopher Belica ◽  
Chloe Wick ◽  
William L. Brown ◽  
...  
Keyword(s):  
Virus Replication ◽  
High Throughput ◽  
Drug Testing ◽  
Living Cells ◽  
Protease Inhibition ◽  
Gain Of Function ◽  
Chemical Methods ◽  
Main Protease ◽  
Viral Polyprotein ◽  
Egfp Fluorescence

AbstractThe main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here we demonstrate a quantitative reporter for Mpro function in living cells, in which protease inhibition by genetic or chemical methods results in strong eGFP fluorescence. This robust gain-of-function system readily distinguishes between inhibitor potencies and can be scaled-up to high-throughput platforms for drug testing.

scholarly journals A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ulrike Frank ◽  
Susanne Kublik ◽  
Dörte Mayer ◽  
Marion Engel ◽  
Michael Schloter ◽  
...  
Keyword(s):  
Cell Death ◽  
Next Generation Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Target Enrichment ◽  
Next Generation ◽  
Mutant Screen ◽  
Specific Target ◽  
Dna Insertion ◽  
Generation Sequencing

Abstract Background Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death. Results Altogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO2 fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants were selected based on visible symptoms. Sensitive mutants showed lesions already after fumigation for 1 h with 10 ppm (ppm) NO2 whereas tolerant mutants were hardly damaged even after treatment with 30 ppm NO2. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. The targeted genome sequencing was highly efficient due to (1.) combination of the pooled DNA from 124 candidate mutants in only two libraries, (2.) successful target enrichment using T-DNA border-specific 70mer probes, and (3.) stringent filtering of the sequencing reads. Seventy mutated genes were identified by at least 3 sequencing reads. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO2-sensitivity or -tolerance confirming that the screen yielded reliable results. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation. Conclusions The presented NO2 dead-or-alive screen combined with next-generation sequencing after T-DNA-specific target enrichment was highly efficient. Two researchers finished the screen within 3 months. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. The experimental design can be easily adapted to other screening approaches e.g. involving high-throughput treatments with abiotic stressors or phytohormones.

scholarly journals The Adenylyl Cyclase Plays a Regulatory Role in the Morphogenetic Switch from Vegetative to Pathogenic Lifestyle of Fusarium graminearum on Wheat

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e91135 ◽  
Author(s):  
Jörg Bormann ◽  
Marike Johanne Boenisch ◽  
Elena Brückner ◽  
Demet Firat ◽  
Wilhelm Schäfer
Keyword(s):  
Adenylyl Cyclase ◽  
Fusarium Graminearum ◽  
Regulatory Role

scholarly journals A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

2011 ◽  
Vol 16 (3) ◽  
pp. 356-362 ◽  
Author(s):  
Eija Martikkala ◽  
Anita Rozwandowicz-Jansen ◽  
Pekka Hänninen ◽  
Ulla Petäjä-Repo ◽  
Harri Härmä
Keyword(s):  
Energy Transfer ◽  
Adenylyl Cyclase ◽  
High Throughput ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Gpcr Activation ◽  
Label Time

G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory Gs or inhibitory Gi proteins. The reproducibility of the assays was excellent, and Z′ values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the Gs-coupled β2AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.

scholarly journals A Canal-Associated Neuron cAMP signalling pathway that regulates C. elegans larval development

2019 ◽  
Author(s):  
Jason Chien ◽  
Fred W. Wolf ◽  
Sarah Grosche ◽  
Nebeyu Yosef ◽  
Gian Garriga ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Larval Development ◽  
Laser Microsurgery ◽  
Gain Of Function ◽  
Specific Expression ◽  
C Elegans ◽  
Similar Phenotype ◽  
Pka Pathway

ABSTRACTCaenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29. ACY-2 is predominantly expressed in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose that cAMP produced by ACY-2 in the CANs acts in neighboring neurons and hypodermal cells where it activates PKA and inhibits KIN-29 to promote larval development. We discuss how this conserved pathway could be partitioned between two cells.

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