TABLE 2

Self-association tests for Gal80 variants

Original Gal4 nonbindersMutant vs. mutantMutant vs. WT
G310D+++++
L319P++++
R189G+++++
G152D+++++
G183S++++
G282S+++++
H261R+++++
A309T++++
D260G
Original Gal3 nonbindersMutant vs. mutantMutant vs. WT
V275E++
V144D++
L406P++
V352E++++++++
D404G++
Y369N++
Controls
DBGal80 WT++++++++
Vector
  • Gal80 single-amino-acid substitution variants were tested for self-association and for association with the wild-type Gal80 in the standard yeast two-hybrid assay. Each variant was reconstituted as a prey construct, VP16Gal80, and tested in yeast ScVP2-103 (SPAL10::URA, GAL1::lacZ gal4Δ, gal80Δ, gal3Δ) for interaction with itself and with the wild-type Gal80, respectively, as baits (DBGal80). As well, we tested each Gal80 variant as a prey, VP16Gal80, for interaction with the wild-type Gal80 as bait, DBGal80. Yeast transformants containing bait and prey plasmids were spotted, with 10-fold dilutions, onto leu trp d.o. for LacZ reporter or leu trp ura d.o. for URA3 reporter. Synthetic media agar plates containing 2% glucose and 1% galactose were used.