TABLE 1.

Controls used in the yeast two-hybrid assay and phenotypes of reporter genes for each control

Four control plasmids, pEXP32/Krev1, pEXP22/RalGDS-WT, pEXP22/RalGDS-m1, and pEXP22/RalGDS-m2, were used to bracket the affinity of an unknown interacting protein. The positive control is based on the interaction of Krev1 and RalGDS protein. Mutation of the RalGDS protein diminishes (RalGDS-m1) or breaks (RalGDS-m2) the interaction with the Krev1 protein. The backbone of pEXP32/Krev1 is a pDEST32 vector containing the GAL4 binding domain coding gene, whereas the backbone of pEXP22/RalGDS-WT, pEXP22/RalGDS-m1, and pEXP22/RalGDS-m2 is a pDEST22 vector containing the GAL4 activation domain coding gene. The empty vectors pDEST32 and pDEST22 were used as the negative controls for vector self-activation. The plasmid pair of pDEST32-FL and pDEST22 was used to test self-activation of the bait pDEST32-FL. Positive interaction in yeast MaV203 cells activates the transcription of three gene reporters (lacZ, HIS3, and URA3). The lacZ gene expresses β-galactosidase, which generates blue coloration in the X-gal assay. Induction of the HIS3 gene allows the yeast transformants to grow on histidine dropout (null) plates. Induction of the URA3 gene endows the yeast transformants the ability to grow on uracil dropout (null) plates, with growth blocked on 5-FOA-containing plates. The abbreviations used are as follows: β-Gal, β-galactosidase; His−, histidine auxotrophy; Ura−, uracil auxotrophy; 5-FOA, 5-fluoroorotic acid; +, growth; −, no growth.

Controls		Purpose	Phenotypes
GAL4 BD plasmid	GAL4 AD plasmid		β-Gal	His−	Ura−	5-FOA
pEXP32/Krev1	pEXP22/RalGDS-WT	Strong positive	Blue	+	+	−
pEXP32/Krev1	pEXP22/RalGDS-m1	Weak positive	Faint blue	+	+	−
pEXP32/Krev1	pEXP22/RalGDS-m2	Negative	White	−	−	+
pDEST32	pDEST22	Negative self-activation	White	−	−	+
pDEST32-FL	pDEST22	Test of self-activation	White	−	−	+