Removing auto-activators from yeast-two-hybrid assays by conditional negative selection

Abstract Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein–protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challe...

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Autores principales:	Devendra Shivhare, Magdalena Musialak-Lange, Irene Julca, Pawel Gluza, Marek Mutwil
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5e8525fb58be461fad6a353f30568090
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spelling	oai:doaj.org-article:5e8525fb58be461fad6a353f305680902021-12-02T15:54:02ZRemoving auto-activators from yeast-two-hybrid assays by conditional negative selection10.1038/s41598-021-84608-92045-2322https://doaj.org/article/5e8525fb58be461fad6a353f305680902021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84608-9https://doaj.org/toc/2045-2322Abstract Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein–protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challenges of yeast two-hybrid assay is the large amount of false-positive hits caused by auto-activators (AAs), which are proteins that activate the reporter genes without the presence of an interacting protein partner. Here, we have developed a negative selection to minimize these auto-activators by integrating the pGAL2-URA3 fragment into the yeast genome. Upon activation of the pGAL2 promoter by an AA, yeast cells expressing URA3 cannot grow in media supplemented with 5-Fluoroorotic acid (5-FOA). Hence, we selectively inhibit the growth of yeast cells expressing auto-activators and thus minimizing the amount of false-positive hits. Here, we have demonstrated that auto-activators can be successfully removed from a Marchantia polymorpha cDNA library using pGAL2-URA3 and 5-FOA treatment, in liquid and solid-grown cultures. Furthermore, since URA3 can also serve as a marker for uracil autotrophy, we propose that our approach is a valuable addition to any large-scale Y2H screen.Devendra ShivhareMagdalena Musialak-LangeIrene JulcaPawel GluzaMarek MutwilNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Devendra Shivhare Magdalena Musialak-Lange Irene Julca Pawel Gluza Marek Mutwil Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
description	Abstract Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein–protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challenges of yeast two-hybrid assay is the large amount of false-positive hits caused by auto-activators (AAs), which are proteins that activate the reporter genes without the presence of an interacting protein partner. Here, we have developed a negative selection to minimize these auto-activators by integrating the pGAL2-URA3 fragment into the yeast genome. Upon activation of the pGAL2 promoter by an AA, yeast cells expressing URA3 cannot grow in media supplemented with 5-Fluoroorotic acid (5-FOA). Hence, we selectively inhibit the growth of yeast cells expressing auto-activators and thus minimizing the amount of false-positive hits. Here, we have demonstrated that auto-activators can be successfully removed from a Marchantia polymorpha cDNA library using pGAL2-URA3 and 5-FOA treatment, in liquid and solid-grown cultures. Furthermore, since URA3 can also serve as a marker for uracil autotrophy, we propose that our approach is a valuable addition to any large-scale Y2H screen.
format	article
author	Devendra Shivhare Magdalena Musialak-Lange Irene Julca Pawel Gluza Marek Mutwil
author_facet	Devendra Shivhare Magdalena Musialak-Lange Irene Julca Pawel Gluza Marek Mutwil
author_sort	Devendra Shivhare
title	Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
title_short	Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
title_full	Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
title_fullStr	Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
title_full_unstemmed	Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
title_sort	removing auto-activators from yeast-two-hybrid assays by conditional negative selection
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/5e8525fb58be461fad6a353f30568090
work_keys_str_mv	AT devendrashivhare removingautoactivatorsfromyeasttwohybridassaysbyconditionalnegativeselection AT magdalenamusialaklange removingautoactivatorsfromyeasttwohybridassaysbyconditionalnegativeselection AT irenejulca removingautoactivatorsfromyeasttwohybridassaysbyconditionalnegativeselection AT pawelgluza removingautoactivatorsfromyeasttwohybridassaysbyconditionalnegativeselection AT marekmutwil removingautoactivatorsfromyeasttwohybridassaysbyconditionalnegativeselection
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Removing auto-activators from yeast-two-hybrid assays by conditional negative selection

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