Spotsizer: High-Throughput Quantitative Analysis of Microbial Growth

Bischof, Leanne; Převorovský, Martin; Rallis, Charalampos; Jeffares, Daniel C.; Arzhaeva, Yulia; Bähler, Jürg

doi:10.2144/000114459

Cited by 12 publications

(16 citation statements)

References 23 publications

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“…Colony size was used as a proxy for double/triple mutant fitness. Colony size measurements were obtained using the gitter package in R 21 or the Spotsizer tool 22 23 . In order to exclude potentially spurious interaction values as a result of linkage, library mutants which fell within 100 kb distance of query mutations were excluded from the respective datasets.…”

Section: Methodsmentioning

confidence: 99%

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

Rallis

Townsend

Bähler

2017

Sci Rep

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The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast.

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Section: Methodsmentioning

confidence: 99%

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

Rallis

Townsend

Bähler

2017

Sci Rep

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“…Unlike most existing methods for quantifying yeast growth on agar plates, our protocol does not measure the size of the individual yeast colonies ( Bischof et al., 2016 ). It instead relies on measuring the overall growth coverage within a given spot.…”

Section: Limitationsmentioning

confidence: 99%

A Quantitative Imaging-Based Protocol for Yeast Growth and Survival on Agar Plates

et al. 2020

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Summary We present a detailed protocol that describes the evaluation of the growth and survival of yeast cells by quantitatively analyzing spotting assays. This simple method reproducibly detects and quantifies subtle differences in growth by measuring the density of cells within a single spot of defined size on an image of a spotting assay. Our protocol is tailored specifically for low-throughput applications, can be easily adapted for specific experimental conditions, and is accessible to yeast experts and non-experts alike. For an example of the execution of this protocol, please refer to DiGregorio et al. (Di Gregorio et al., 2020).

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“…5a ). Images on YES control plates and YES-formamide were obtained and analysed using Spotsizer – an automated image analysis platform for colony size measurement 39 . This software standardizes every colony area by the median of its plate and it calculates the ratio between control and experimental condition for each deletion (raw data is provided in Supplementary File 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Automated colony size ratio for genome-wide formamide sensitivity screen was performed using Spotsizer open software(ref. 39 ; http://doi.org/10.4225/08/57315CD446E2C ). GO enrichment analysis was performed by online AnGeLi software(ref.…”

Section: Methodsmentioning

confidence: 99%

RNA metabolism is the primary target of formamide in vivo

Hoyos-Manchado

Reyes-Martín

Rallis

et al. 2017

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The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes.

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Spotsizer: High-Throughput Quantitative Analysis of Microbial Growth

Cited by 12 publications

References 23 publications

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

A Quantitative Imaging-Based Protocol for Yeast Growth and Survival on Agar Plates

RNA metabolism is the primary target of formamide in vivo

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