Table 5.
Two-hybrid interactions between Spc110p and components of the Tub4p complex
| Gal4p-DNA binding domain fused to | Gal4p-activation domain fused to | Growth on [-trp,-leu,-ade] platesa | Colony colorb |
|---|---|---|---|
| A. wt proteins | |||
| Spc98p | Spc110p (aa 1-183) | ++++ | Bc |
| Spc97p | Spc110p (aa 1-183) | − | Bd |
| Tub4p | Spc110p (aa 1-183) | −/+ | LB (≤1%) |
| Spc98p | Spc110p (aa 1-150) | +++ | B |
| Spc97p | Spc110p (aa 1-150) | − | W (3d); B (5d)e |
| B. Mutant proteins | |||
| Spc98p | Spc110-221p (aa 1-183) | ++ | W |
| Spc97p | Spc110-221p (aa 1-183) | nd | W |
| Spc98-63p | Spc110p (aa 1-183) | ++++ | B |
| Spc98-63p | Spc110p (aa 1-150) | ++ | nd |
| Spc98-63p | Spc110-221p (aa 1-183) | − | nd |
| Spc97-62p | Spc110p (aa 1-183) | − | W |
| Spc97-114p | Spc110p (aa 1-183) | − | W |
| C. Controls | |||
| laminf | Spc110p (aa 1-183) | − | W |
| lamin | Spc110p (aa 1-150) | − | W |
| lamin | Spc110-221p (aa 1-183) | − | W |
Growth of cells on SD-trp, -leu, -ade (−TLA) plates is scored between day 3 and when colonies are fully developed by day 5 or 6. When colonies are fully developed, the plating efficiency on −TLA plates is compared to the plating efficiency on SD-trp, -leu plates (−TL). ++++, plating efficiency on −TLA is ≥90% of −TL; colonies are fully developed on day 3; +++, ≥90% plating of −TL, colonies are small on day 3 and fully developed by day 5; ++, 50-90% plating of −TL; colonies are barely visible on day 3, fully developed by day 5; +, 50% plating of −TL; colonies are barely visible on day 3, fully developed by day 6; +/−, 25% plating of −TL; colonies are not visible on day 3, fully developed by day 6; −/+, no growth on day 3; colonies are barely visible on day 5, but do not get bigger thereafter; −, no growth. Results are tabulated from at least two independent transformations.
Colony-lift filter assays using X-gal are performed to measure β-galactosidase activity. (%) indicates the % of the total number of colonies transferred onto nitrocellulose filters that display color. A (≤5%) is considered background rather than a true interaction that occurs when 50-95% of transferred colonies display color after a 30° incubation overnight. Generally, “blue” colonies are blue within the first 1–3 h after incubation, but the final scoring reported here is for an overnight incubation to include weak interactions. B, blue; LB, light blue; W, white; nd, not determined. Observations are made from at least two independent transformations.
Blue colonies develop within 1 h of incubation at 30°C.
Blue colonies develop after 2 h incubation at 30°C.
Assays were performed using 3- or 5-d-old transformants.
Lamin instead of the vector pAS2 was used as the negative control because pAS2 by itself activates transcription of the reporter genes.