Table 1.
CBP interacts with DAF-16 in yeast and mammalian two-hybrid system
| Yeast two-hybrid system
|
Mammalian two-hybrid system
|
||||||
|---|---|---|---|---|---|---|---|
| Clone | Amino acids | No. of clones | lacZ+/HIS | GAL4⋅DAF-16 derivative | Activity (Luc/β-gal)
|
-Fold effect of p300 | |
| VP16 | VP16·p300 | ||||||
| DAF-16 | 176–508 | 1 | ++/+ | GAL4⋅BD | 0.9 ± 0.09 | 0.91 ± 0.03 | 1 |
| F38A6.3 (HIF-1α) | 104–343 | 5 | ++/+ | GAL4⋅DAF-16 wild type | 31 ± 1.89 | 284 ± 5.03 | 9 |
| T01B10.4 (HNF4) | 51–450 | 2 | +/+ | GAL4⋅DAF-16 4(S/T-A) | 57 ± 2.23 | 328 ± 22.4 | 6 |
| ZK1290.4 (NF-1) | 525–1026 | 1 | ++/+ | GAL4⋅DAF-16 (Δ340–511) | 4.5 ± 0.14 | 12.5 ± 0.93 | 3 |
Yeast two-hybrid system: Two million independent colonies were screened and 92 positive clones were isolated by using the reporter genes lacZ and HIS3. Yeast plasmids encoding the 92 “preys” were rescued into Escherichia coli HB101 and used in new yeast transformation experiments to confirm the two-hybrid interaction. On the second round of screening 46 clones were positive with GAL4⋅CBP-1, but not with the GAL4 DNA-binding domain alone or GAL4LAM5′-1 (CLONTECH). Twenty-two of the 46 clones encoded putative C. elegans transcription factors, some of which have known mammalian homologs. A partial list is shown in this table. The predicted open reading frames (ORFs) of clones F38A6.3, T01B10.4, and ZK1290.4 encode proteins related to mammalian HIF-1α, HNF4, and NF-1, respectively. The length of the ORFs of the isolated clones, the number of independent clones identified for each interacting molecule, and the relative strength of the interactions is shown in columns 2, 3, and 4, respectively. Mammalian two-hybrid system: GAL4⋅DAF-16 derivatives (2 μg) were cotransfected into HEK 293 cells with VP-16 alone or VP-16⋅p300 (2.5 μg) and the luciferase (15 μg) or β-galactosidase (2.5 μg) reporter genes as described in the text. Cells were assayed for luciferase (Luc) and β-galactosidase (β-gal) activity. Luciferase activity was corrected for coexpression of β-galactosidase activity (±SEM). The fold effect indicates the specific interaction of the GAL4⋅DAF-16 derivative with VP-16⋅p300 compared to VP-16 alone.