Activity of a single copy of the new mad2 alleles. mad2+ (#) indicates the normal wild-type mad2 allele, while mad2+, mad2-56, and mad2-64 indicate that these alleles were replaced with the mad2Δ allele together with the kanr marker gene (see materials and methods). mad2Δ denotes the mad2 allele disrupted by insertion of the ura4+ gene, the kanr marker was not used here. (A) TBZ sensitivity test. YE containing 20 μg/ml TBZ was used together with a control plate. Incubation was at 33° for 3 days. Strains used were HM123, HM713, YT547, YT549, and YT553. (B) Activity of mitotic arrest. Strains used were the same as in A. CBZ (50 μg/ml) was added to a logarithmic-phase culture and incubated for the indicated time periods. Percentage of cells containing hypercondensed chromosomes was measured. mad2+(#) (solid circles); mad2+ (open circles); mad2Δ (crosses); mad2-56 (open triangles); mad2-64 (open squares). More than 200 cells were scored for each strain. (C) Capability of different mad2 alleles to support the growth of gtb1 mutant. Incubation was at 33° or 30° for 3 days. Strains used were W604, YT578, YT555, YT556, and YT559. (D) Patterns of chromosome segregation in late anaphase. Strains with indicated mad2 alleles in the gtb1-93 background (same as in C) were incubated at 30° in YE for 1.5 hr after the treatment with HU and stained with DAPI. More than 170 mitoses were scored for each strain.