A novel genetic system to detect protein–protein interactions

  title={A novel genetic system to detect protein–protein interactions},
  author={Stanley Fields and Ok‐kyu Song},
PROTEIN-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization1. It consists of… 

Yeast two-hybrid assay for studying protein-protein interactions.

  • A. Osman
  • Biology
    Methods in molecular biology
  • 2004
This chapter presents an outline for the GAL4-based yeast two-hybrid system with a detailed description of the vectors, host cells, and methods for detection and verifying protein interactions.

Using the yeast two-hybrid system to identify protein-protein interactions.

Protocols to test interactions between two individual proteins and to look for novel interacting partners by screening a single protein or domain against a library of other proteins using a GAL4 based yeast two-hybrid system are described.

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli

A new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli is developed, based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain.

Generation of a yeast two-hybrid strain suitable for competitive protein binding analysis.

The yeast two-hybrid (Y2H) system has been broadened and applied for further characterization of protein-protein interactions, and the URA3 gene is replaced in the standard strain L40c, aimed to create a strain still exploitable for Y2H screens, but additionally allowing the simultaneous expression of a third protein.

Mapping Protein-Protein Interaction Using High-Throughput Yeast 2-Hybrid.

This chapter provides a step-by-step protocol for the Y2H experimentation preceded by a materials listing while simultaneously including key notes throughout the entire experimental process of biological-mechanistic and historical understandings of the steps.

Identification of interacting proteins using the yeast two-hybrid screen.

A two-hybrid yeast system has been used to successfully characterize protein interactions among signaling molecules and can be used to identify new binding partners for a protein of interest or define the interaction domains and relative affinity between two proteins known to interact.



A yeast gene that is essential for release from glucose repression encodes a protein kinase.

Findings indicate that SNF1 encodes a protein kinase and suggest that protein phosphorylation plays a critical role in regulation by carbon catabolite repression in eukaryotic cells.

Primary structure of the Saccharomyces cerevisiae GAL4 gene

The GAL4 gene encodes a positive regulator of the galactose-inducible genes in Saccharomyces cerevisiae and its 2.8-kilobase mRNA has been identified, and the DNA sequence and the mapping of the 5' and 3' ends of its transcripts are reported.

Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization.

Study of intracellular compartmentalization in yeast of Escherichia coli beta-galactosidase bearing heterologous amino acid sequences at its amino terminus shows results consistent with the proposal that the GAL4 gene product mediates positive control by binding to DNA and that the information for nuclear localization resides in its aminoterminus.

Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein.

These and related findings support the idea that GAL4 activates transcription by touching other DNA-bound proteins.

Binding of the virion protein mediating alpha gene induction in herpes simplex virus 1-infected cells to its cis site requires cellular proteins.

It is reported that alpha-trans-induction factor, synthesized in vitro or present in nuclear extracts of infected cells, forms complexes with viral DNA fragments containing its cis-acting site only in the presence of cellular proteins and only under conditions that also enable the binding of the alpha H1 protein to the DNA.

Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL 10-CYC1-lacZ fusion was constructed in vitro and defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction.

Transformation of yeast.

This work has used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced.

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation

TheCDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids and may contain genes that compensate for the lack of CDC8 gene product.