Fig. 6. The C–terminal 20 amino acid residues of Cdc27 are required for in vivo function. (A) pREP3X plasmids expressing the indicated mutant proteins (note that FFAA = F368AF369A) were transformed into cdc27⁺/cdc27Δ diploid cells. Transformant colonies were then sporulated and spores prepared by helicase treatment plated onto EMM medium as described in the text. After 3–4 days incubation at 30°C, the degree of rescue of the cdc27Δ phenotype was scored subjectively as follows: +++, excellent rescue, cell size at cell division equivalent to wild type; ++, good rescue, cell size at cell division typically in the range 16–20 μm compared with 14 μm for cells expressing the wild-type Cdc27; +, weak rescue, cell size at division typically >20 μm; –, no rescue, indistinguishable from cells transformed with pREP3X vector. (B) cdc27Δ cells expressing Cdc27-1–362 or Cdc27-F368A/F368A (FFAA) grown by necessity in EMM medium lacking thiamine were plated onto EMM medium containing either HU or MMS at the concentrations indicated and scored for growth after 4–6 days.