TABLE 2.

Assays for testing protein interactions in the yeast two-hybrid system

Four specific assays with the corresponding metabolic selection plates were used to test the induction of three reporter genes in yeast cells. Reporter gene activation provided four unique phenotypes with which to assess the putative protein interactions: (i) blue colony coloration for the X-gal assay; (ii) growth on the Leu−/Trp−/His− with 80 mm 3AT plates; (iii) growth on the Leu−/Trp−/Ura− plates; and (iv) failure to grow on Leu−/Trp− with 0.2 weight % 5-FOA plates. The abbreviations used are as follows: β-Gal, β-galactosidase; Leu−/Trp−/His−, auxotrophic plate lacking leucine, tryptophan, and histidine; Leu−/Trp−Ura−, auxotrophic plate lacking leucine, tryptophan, and uracil; Leu−/Trp−, auxotrophic plate lacking leucine and tryptophan; 5-FOA, 5-fluoroorotic acid.

Test	Assay	Plates	Phenotypes
			Interaction	No interaction
lacZ induction (β-Gal activity)	X-gal assay	YPAD containing filter paper	Blue	White
HIS3 induction	Histidine auxotrophy	Leu − /Trp − /His − containing 80 mm 3AT	Formed colony	No colony
URA3 induction	Uracil auxotrophy	Leu−/Trp−/Ura−	Formed colony	No colony
URA3 induction	5-FOA sensitivity	Leu − /Trp − containing 0.2 weight % 5-FOA	No colony	Formed colony