Table 2.
Ability of mid1 mutant constructs to complement mid1 and plo1 mutants
| Strains | Complementation (% abnormal
septa)
|
Localization | |
|---|---|---|---|
| 30°C | 36°C | ||
| wt | NA (0.5) | NA (1.5) | Nucleus/broad band tight ring |
| mid1-Δ strain | NA (98.5) | NA (99.8) | NA |
| ΔNES1-mid1 | + (20.5) | − (75.8) | Nucleus + occasional faint rings |
| ΔNES2-mid1 | ++ (1.0) | − (79.0)a | Nucleus + faint rings |
| ΔNES1+2-mid1 | − (74.6) | − (98.9)a | Nucleus + occasional faint rings |
| NLS*-mid1 | ++ (0.5) | ++ (1.0) | Broad band tight ring |
| ΔPH-mid1 | ++ (nd) | ++ (4.0) | Nucleus/broad band tight ring |
| mid1 1-506 | ++ (nd) | ± (53.6) | Diffuse punctuate staining |
| 25°C | 36°C | ||
| plo1-1 + pRep 41x | NA (1.8) | − (94.8) | |
| plo1-1 + NLS*-mid1 | NA (2.5) | − (95.5) | |
Wild type strain, mid1Δ strain and mid1Δ strain carrying integrated constructs expressing mid1 mutants were grown at 30°C or 36°C, stained with calcofluor, and assayed for septation defects by microscopic analysis. Plo1-1 strain with vector pREP41x only or an integrated copy of NLS*-mid1 construct were grown in minimal medium at 25°C or shifted from 25°C to 36°C for 4 h. Mean percentages of cells with abnormal septa were calculated from two separate experiments using two independent clones (n > 200 cells with a septum). NA, not applicable; nd, not determined.
Functionality in these mutants was difficult to assess because Western blot analysis showed that these constructs were expressed at a lower level than wild-type mid1p at 36°C (see Figure 7).