is required for Ama1 degradation during meiosis. A: Wild-type (RSY335), cdc20-1 (RSY809) and cdc16-1 strains (RSY954) harboring Ama1p-T7 (pKC3036) were induced to enter the meiosis and timepoints taken as indicated. Immunoblot analysis of immunoprecipitated protein extracts was conducted to detect Ama1p. Immunoblot analysis of Tub1p was used as a loading control. MI and MII indicate the approximate times of meiosis I (MI) and meiosis II (MII) as determined by DAPI analysis. All the strains were grown at 23°C and switched to 34.5°C (restrictive temperature for both cdc20-1 and cdc16-1 strains) after 4.5 h at 23°C in SPM. B: Quantitation of Ama1p-T7 from the experiments conducted in A. C: The levels of Ama1p-T7 were monitored in a cdc20-1 strain as in Panel A except that the cells were switched to the restrictive temperature 15 h after transfer to SPM. This panel also contains analysis of Ama1p-T7 stability at both temperatures, 30 h after entering sporulation. D: As in Panel A except that the wild type (RSY335) and cdc20-1 (RSY809) cultures harbored either Clb5p-3HA (pKC440) or Clb1p-9HA (pKC427) expression plasmids.