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Fig. 7. (A) N-terminus 77 amino acids are important in augmenting centromeric cohesion. cdc20 slk19 GAL-CDC20 strains carrying TetO/GFP-TetR constructs transformed with a CEN plasmid expressing full-length Slk19, Slk19-R77E or Slk19-(1-77) (US3986, US4024, US4025) were arrested in G2-M by growth in YEPD to allow spindle formation and transient separation of centromeres. The culture was split into two halves: nocodazole was added to one half for 1.5 hours to destroy spindles, while the other half continued without Nocodazole. Samples were collected and cells (150 cells were counted) with separated centromeres were quantitated. (B) Coiled-coil domains in C-terminus of Slk19 are important for physical association with Scc1. Extracts prepared from asynchronously growing WT strains with endogenously tagged SCC1-HA₆ and carrying either SLK19-cmyc₁₂, slk19-(327-400)-cmyc₁₂, slk19-(419-498)-cmyc₁₂, slk19-(511-538)-cmyc₁₂ or slk19-(543-577)-cmyc₁₂ on CEN plasmid (US4532, US4022, US4020, US4021, US4023) were immunoprecipitated with anti-HA or anti-cmyc beads. Immunoprecipitates were analyzed by western blotting using anti-cmyc or anti-HA antibodies respectively. Extracts from untagged WT (US1363) and WT cells with endogenously tagged SCC1-HA₆ and carrying SLK19-cmyc₁₂ on a plasmid (US4532) were used as negative and positive controls. Table: Slk19 constructs carrying deletions in the coiled coil domains are unable to restore centromeric elasticity. cdc20 slk19 cells carrying TetO/GFP-TetR constructs and transformed with various deletions in the coiled coil domains of Slk19 (US4067, US4070 and US4071) were arrested in G2. Samples were collected and proportion of cells (150 cells counted) with divided centromeres was determined.

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