TABLE 1.
Interaction of B. subtilis Dna initiation protein analyzed by the yeast two-hybrid system
| Protein fused to Gal4 BD | X-Gal assay (β-galactosidase activity)a
|
|||||
|---|---|---|---|---|---|---|
| Protein fused to Gal4 AD
| ||||||
| None | DnaA | DnaB | DnaC | DnaD | DnaI | |
| No | − (0) | − (71 ± 48) | − (0) | − (0) | − (0) | − (0) |
| DnaA | − (0) | − (0) | − (80 ± 64) | − (0) | − (23 ± 1) | − (0) |
| DnaB | − (25 ± 18) | − (0) | + (720 ± 30) | − (0) | − (0) | − (0) |
| DnaD | − (180 ± 125) | + (2,467 ± 80) | − (0) | − (0) | + (5,555 ± 391) | − (0) |
| DnaD23 | NDb | − (151 ± 19) | ND | ND | + (4,219 ± 305) | ND |
+, yeast colonies showed a blue color within 4 h under the assay conditions; −, still white even after 8 h of incubation. In each combination, five independent clones were used for this assay and all showed the same result (data not shown). X-Gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. The enzyme activity is represented as units in 1 ml of cell suspension with an optical density of 1.0 at 600 nm. One unit defines the amount of enzyme that hydrolyzes 1 pmol of MUG (4-methylumbelliferyl-β-d-galactoside) per min. Average units from three independent experiments and their standard errors are shown. As shown previously (8), BD-DnaC and BD-DnaI interacted with AD-DnaI and AD-DnaC, respectively, indicating that the latter two fusion proteins used in this study are active in yeast cells.
ND, not done.