The yeast two-hybrid screen is a genetic assay to detect protein-protein interactions in vivo. This method, developed by Prof. Stan Fields and coworkers (1) can be used to investigate the interaction of two known proteins (e.g. defining domains or amino acids critical for interactions). Another important application is the identification of
previously unknown binding partners of specific proteins by screening a
library.

Positive clones are selected by their ability to activate the transcription of reporter genes which enable the auxotrophic yeast two-hybrid strains to grow on nutritionally selective media. Most two-hybrid methods also use the E. coli lacZ gene as a second reporter gene. Usually the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ in a second screen by a time-consuming filter-lift assay. Alternatively, in yeast strains producing a-galactosidase the chromogenic substrate X-a-Gal can be used to assay GAL4-based two-hybrid interactions directly on the nutritional selection plates (2).

X-a-Gal is a chromogenic substrate for a-galactosidase (also known as
melibiase or alpha-D-galactoside galactohydrolase, EC 3.2.1.22), an enzyme which enables yeast to use the disaccharide melibiose as a carbon source during growth or fermentation. In the yeast Saccharomyces cerevisiae this enzyme is encoded by the MEL1 gene which is regulated by several GAL genes. Secretion of this enzyme in response to GAL4 activation leads to hydrolysis of X-a-Gal in the medium causing yeast colonies to develop a blue colour. A big advantage of this reporter system is that it abolishes the need for b-galactosidase filter-lift assays and the result can be seen immediately on the selection plates.

Yeast strains which can be used with include the strains AH109, PJ69-2A, Y190, and Y187 sold by Clontech for the "Matchmaker yeast two-hybrid system " (Matchmaker is a trademark of CLONTECH Laboratories, Inc.).

1. S Fields and O.-K. Song, A Novel Genetic System to Detect Protein-Protein Interactions. Nature, 340, 245-246 (1989)
2. S. Aho, A. Arffman, T.Pummi and J. Uitto, A novel reporter gene MEL1 for the yeast two-hybrid system. Anal. Biochem., 253, 270-272 (1997)

How to make X-a-Gal Indicator Plates

To make X-a-Gal indicator plates, X-a-Gal can be either spread on top of the medium or included in the medium prior to pouring the plates.

To spread X-a-Gal onto pre-made plates:
If using freshly poured plates, allow yeast dropout medium to cool down and harden at room temperature. Spread 200-500 ml of X-a-Gal stock solution (2 mg/ml in DMF) onto a 15-cm plate or 100-200 ml onto a 10-cm plate evenly. Leave plates to dry at room temperature. Plate cells and incubate at 30°C until blue colonies form.

To pour X-a-Gal indicator plates:
Autoclave the appropriate dropout agar medium. Let it cool to 55°C before adding 1 ml of X-a-Gal stock solution (20 mg/ml in DMF) per 1 litre medium. Pour plates and allow medium to harden at room temperature. Plate cells and incubate at 30°C until blue colonies form.

Stock solutions of X-a-Gal should be stable for at least 6 months if kept in the dark at -20°C.

A colony-colour method which differentiates a-galactosidase positive strains of yeast.

R.S. Tubb and P.L. Liljestrom, J. Inst. Brew. 92, 588, (1986)

Histochemical detection of a-D-galactosidase with 5-Br-4-Cl-3-indoxyl a-D-galactoside

R. Gossrau and Z. Lojda, Acta histochem., 85, 213 (1989)

X-a-gal-based medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk.

P. Chevalier, D. Roy and and L. Savoie, J.Microbiol. Meth., 13, 75 (1991)

American Society of Brewing Chemists. Report of Subcommittee on X-a-gal medium.

J. Amer. Soc. Brew. Chem., 51, 185, (1993)