Figures & data
Fig. 1. Isolated esp1-ts mutants are defective in sister chromatid disjunction.
Notes: (A) Separase promotes both anaphase onset and early anaphase progression. Upon anaphase onset, separase cleavages cohesin and promotes sister chromatid separation. Thereafter, separase stimulates hyper-condensation of the highly repeated rDNA (coding rRNA) via the Cdc14 early anaphase release (FEAR) pathway in a protease-independent manner, promoting segregation of the long chromosome XII (Chr XII) harboring rDNA. Protease-deficient separase mutations cause metaphase arrest without sister chromatid separation, while FEAR-deficient separase mutations should cause early anaphase arrest with segregated nuclei. (B) Ten-fold serial dilutions of cells of esp1-ts mutants were spotted in 1 µl drops onto YPAD plates, and the plates were incubated at 25 or 37 °C for 1 day. (C) esp1-ts cells have large buds with undivided nuclei and some cells have rebuds at restrictive temperatures. Cells grown at 25 °C were transferred to 37 °C overnight and images of DAPI-stained nuclei were observed.
Fig. 2. Some esp1-ts are hypersensitive to microtubule and DNA damage.
Notes: Ten-fold serial dilutions of cells of esp1-ts strains were spotted in 1 μl drops onto YPAD plates in the presence or absence of the microtubule poison benomyl (15 μg/ml) or the DNA-damaging agent bleomycin (2 μg/ml) and the plates were incubated at 30 °C for 1 day.
Table 1. Summary of phenotypes of esp1-ts mutants.
Fig. 3. Carbon-poor conditions alleviate the temperature sensitivities of some esp1-ts mutants.
Notes: Fivefold serial dilutions of cells of esp1-ts strains were spotted in 1 μl drops onto three synthetic medium plates containing different carbon and nitrogen sources: control (2% glucose and 0.5% ammonium sulfate), carbon poor (3% glycerol and 0.5% ammonium sulfate), and nitrogen poor (2% glucose and 0.1% proline). The plates were incubated at 34 °C for 2 days for control and carbon-poor plates and for 3 days for nitrogen-poor plates.
Fig. 4. Osmotic stress alleviates the temperature sensitivities of some esp1-ts mutants.
Notes: Fivefold serial dilutions of cells of esp1-ts strains were spotted in 1 μl drops onto YPAD plates in the presence or absence of 1.2 M sorbitol and the plates were incubated at 34 °C for 2 days.
Fig. 5. Effects of overexpression of securin on esp1-ts mutants.
Notes: Ten-fold serial dilutions of cells of esp1-ts strains harboring plasmid pSCU974 (PDS1 2μ URA3)Citation29) or pSCU154 (pRS426; empty vector, EV) were spotted in 1 μl drops onto SD-Ura plates. The plates were incubated at 30 °C for 1 day.
Fig. 6 Effects of overexpression of MPT5 on esp1-ts mutants.
Notes: Ten-fold serial dilutions of cells of esp1-ts strains harboring plasmid pSCU1079 (MPT5 2μ URA3)Citation30) or pSCU154 (pRS426; empty vector) were spotted in 1 μl drops onto SD-Ura plates. The plates were incubated at 30 °C for 1 day. Note that esp1-304 transformants with the plasmid overexpressing MPT5 could be obtained.
Supplemental material