Please use this identifier to cite or link to this item:
Title: Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
Authors: Shivhare, Devendra
Musialak-Lange, Magdalena
Julca, Irene
Gluza, Pawel
Mutwil, Marek
Keywords: Science::Biological sciences
Issue Date: 2021
Source: 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(1), 5477-.
Project: MOE2018-T2-2-053
Journal: Scientific Reports
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.
ISSN: 2045-2322
DOI: 10.1038/s41598-021-84608-9
Schools: School of Biological Sciences 
Rights: © 2021 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

Files in This Item:
File Description SizeFormat 
s41598-021-84608-9.pdf1.42 MBAdobe PDFThumbnail

Citations 50

Updated on Oct 1, 2023

Web of ScienceTM
Citations 50

Updated on Sep 30, 2023

Page view(s)

Updated on Oct 3, 2023

Download(s) 50

Updated on Oct 3, 2023

Google ScholarTM




Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.