Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast.

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• The yeast one-hybrid (Y1H) system is crucial for identifying DNA-protein interactions.
• Using full gene promoters in Y1H assays can reduce accuracy and sensitivity, particularly in complex organisms.
• Efficient identification of suitable promoter fragments is essential for reliable Y1H results.

Purpose of the Study:

• To develop a robust workflow for identifying conserved, biologically relevant promoter fragments for Y1H assays.
• To establish efficient high-throughput screening methods for DNA-protein interaction studies.
• To facilitate the construction of gene regulatory networks.

Main Methods:

• Phylogenetic analysis to identify conserved promoter fragments in Arabidopsis thaliana.
• Manual and automated (robotized) high-throughput Y1H screening of arrayed open reading frame (ORF) libraries.
• Application of the method for yeast two-hybrid (Y2H) screenings.

Main Results:

• A workflow for selecting optimal DNA baits for Y1H screenings was established.
• Efficient high-throughput screening protocols, including an automated version, were detailed.
• The described methods are scalable and applicable to Y2H screenings for building gene regulatory networks.

Conclusions:

• The developed workflow enhances the identification of DNA-protein interactions using Y1H assays.
• Phylogenetic analysis combined with high-throughput screening provides a powerful approach for selecting promoter fragments.
• This methodology supports the comprehensive study of gene regulation and network construction.