TABLE 2.
Confirmation of specific menin-RPA2 interaction in a yeast two-hybrid assay based on transactivation of a Gal4-regulated lacZ reporter genea
| Proteinb | β-Galactosidase activity
|
||
|---|---|---|---|
| Gal4 DBD | Menin | p53* | |
| Gal4 AD | − | − | − |
| RPA2 (59) | − | ++ | ND |
| RPA2 (70) | − | ++ | ND |
| RPA2 (coding sequence) | − | ++ | ND |
| JunD* | − | +++ | − |
| RPA1 | − | − | ND |
| RPA3 | − | − | ND |
| RPA4 | − | − | ND |
| TAg* | − | − | ++++ |
Yeast strain Y190, which contains a genomic Gal4-regulated lacZ reporter gene, was transformed with plasmids expressing the indicated bait and prey proteins and scored for β-galactosidase activity in a colony filter lift assay after growth on CM/glucose medium lacking leucine and tryptophan at 30°C. ND, not determined; ++, +++, and ++++, moderate, high, and very high levels of β-galactosidase activity, respectively; −, no activity. The bait proteins menin, p53* (amino acids 72 to 390 of mouse p53), RPA1, RPA3, and XPA were fused at their N termini to the S. cerevisiae Gal4 DNA-binding domain (DBD). The prey proteins RPA2, JunD* (amino acids 8 to 340 of human JunD), RPA1, RPA3, RPA4, and TAg* (amino acids 87 to 708 of simian virus 40 T antigen) were fused at their N termini to the S. cerevisiae Gal4 activation domain (AD).
As indicated in Table 1, the numbers in parentheses are identifiers for the two independent RPA2 cDNA clones obtained in the initial library screen. Coding sequence refers to the cDNA sequence encoding the full-length (amino acids 1 to 271) RPA2 protein.