Table 2.
Determination of the sequences that interact in human FAS dimer formation by using the yeast two-hybrid system
| GAL4-DB plasmids | GAL4-ACT plasmids | β-Gal Activity |
|---|---|---|
| pAS2 | pACT2 | 2 |
| pAS2-ID | pACT2 | 10 |
| pAS2 | pACT2-ID | 10 |
| pAS2-ID | pACT2-ID | 487 |
| pAS2-ID ← | pACT2-ID ← | 13 |
| pAS2-ID-N | pACT-ID | 92 |
| pAS2-ID-C | pACT2-ID | 182 |
| pAS2-ID-N | pACT2-ID-C | 271 |
| pAS2-ID-N ← | pACT2-ID ← | 13 |
| pAS2-ID-C ← | pAS2-ID-C ← | 13 |
| pAS2-ID-C | pACT2-ACP-TE | 16 |
| pAS2-ID-C | pACT2-KS | 56 |
| pAS2-KS | pACT2-ACP-TE | 18 |
Plasmids pAS2 and pACT2 are the vectors that contain sequences coding for the DNA-binding and activation domains (GAL4-DB and GAL4-ACT) to which the FAS sequences are fused in-frame at their C-terminal ends. The plasmid constructs used in these experiments are described in Fig. 2 and in Materials and Methods. The left arrow (←) indicates that the cDNA sequences coding for ID are fused in the opposite orientation to either the GAL4-DB or the GAL4-ACT domain and hence do not code for the ID sequences. These constructs are not described in Fig. 2. β-Galactosidase (β-Gal) activity is measured in permeabilized yeast cell suspensions by using O-nitrophenyl-β-d-galactoside as substrate. The formed O-nitrophenol was measured at 420 nm, and the optical density of cell suspension was measured at 600 nm. One unit of β-Gal activity equals 0.001 OD420/OD600/min. The average values of three independent transformants are shown. The variation among replicates is <5%.