TABLE 4.
plc1Δ extracts exhibit reduced MT binding to minichromosomes
| MT concn (MTs/ml) | Avg % of minichromosomes in the MT pelleta
|
|||||
|---|---|---|---|---|---|---|
| Asynchronous cells
|
Nocodazole-arrested cells
|
|||||
| Wild type
|
plc1Δ
|
Wild-type CEN | plc1Δ CEN | |||
| CEN | 2μm | CEN | 2μm | |||
| 0 | 2.1 ± 0.3 | 2.0 ± 0.4 | 2.5 ± 0.6 | 2.6 ± 0.6 | 2.0 ± 0.3 | 1.9 ± 0.3 |
| 4 × 107 | 8.7 ± 1.5 | 2.5 ± 0.5 | 5.1 ± 1.0 | 2.3 ± 0.4 | 19.9 ± 2.7 | 12.6 ± 2.1 |
| 4 × 108 | 21.3 ± 2.9 | 3.1 ± 0.5 | 10.9 ± 2.0 | 2.9 ± 0.5 | 43.5 ± 6.9 | 25.2 ± 4.6 |
| 4 × 109 | 24.2 ± 3.8 | 3.8 ± 0.6 | 13.4 ± 2.1 | 3.7 ± 0.6 | 47.7 ± 7.8 | 28.1 ± 4.9 |
Cleared lysates were prepared from W303-1a and plc1Δ cells (HL1-1) transformed with the centromeric plasmid pRS414 or the 2μm plasmid pRS424. Cells were arrested at the G2/M stage of the cell cycle by incubating them for 6 h in the presence of 15 μg of nocodazole per ml. Under these conditions, 90% of both wild-type and plc1Δ cells arrested as large-budded cells. Different amounts of bovine MTs were added to the lysates and incubated for 15 min at 20°C. MTs were pelleted, and the percentage of minichromosomes that cosedimented with the MTs was determined. The experiment was performed five times, and the standard deviations are indicated. The amount of the 2μm plasmid cosedimenting from lysates of nocodazole-arrested cells did not significantly differ from the amount cosedimenting from lysates of asynchronous cells (data not shown).