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

Shivhare, D., Musialak-Lange, M., Julca, I., Gluza, P., & Mutwil, M. (2021). Removing auto-activators from yeast-two-hybrid assays by conditional negative selection. Scientific Reports, 11: 5477. doi:10.1038/s41598-021-84608-9.

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 Creators:
Shivhare, Devendra1, Author
Musialak-Lange, M.2, Author              
Julca, Irene1, Author
Gluza, P.2, Author              
Mutwil, M.2, Author              
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1external, ou_persistent22              
2Regulatory Networks, Department Stitt, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753332              

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 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.

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Language(s): eng - English
 Dates: 2021
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.1038/s41598-021-84608-9
Other: Shivhare2021
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Title: Scientific Reports
  Abbreviation : Sci. Rep.
Source Genre: Journal
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Publ. Info: London, UK : Nature Publishing Group
Pages: - Volume / Issue: 11 Sequence Number: 5477 Start / End Page: - Identifier: ISSN: 2045-2322
CoNE: https://pure.mpg.de/cone/journals/resource/2045-2322