TABLE 2.
DH-dependent oligomerization of GRF in the yeast two-hybrid systema
| GAL4 transactivation domain fusion | GAL4 DNA-binding domain fusion | Growth in the presence of 3-AT | β-Galactosidase activity |
|---|---|---|---|
| AD-SNF1 | BD-mGRF1(221–497) | − | 0.01 ± 0.002 |
| AD-hGRF2(176–474) | BD-MGRF1(221–497) | + | 0.19 ± 0.02 |
| AD-hGRF2(176–474) | BD-mGRF1(221–497)IIIRDII | + | 0.14 ± 0.04 |
| AD-hGRF2(176–474) | BD-mGRF1(221–497)L263Q | − | ND |
| AD-hGRF2(176–474) | BD-mGRF1(221–460) | + | 0.48 ± 0.04 |
| AD-hGRF2(234–474) | BD-mGRF1(221–497) | + | 0.17 ± 0.02 |
Yeast strain Y190 was cotransformed with a GAL4 transactivation domain plasmid and a GAL4 DNA-binding domain plasmid and analyzed for growth in the absence of His. Cells that grew in the absence of tryptophan, leucine, and histidine but in the presence of 25 mM 3-amino-1, 2, 4-triazole (3-AT) were then tested for β-galactosidase activity (26). AD-hGRF2(234–474) concerns the DH domain without IQ sequences. BD-mGRF1(221–497)IIIRDII and BD-mGRF1(221–497)L263Q concern point mutations in the DH domain as described in the text. BD-mGRF1(221–460) concerns the DH domain without PH sequences. β-Galactosidase activities represent the average for six colonies from two independent transformations. The β-galactosidase activity for the AD-SNF1/BD-mGRF1(221–497) pair was obtained from colonies that grew on an accompanying plate that selected for growth in the absence of tryptophan and leucine only. ND, not determined.