Figure 7.

(A) Analysis of CDE III-binding activity in extracts from glc7-10 (lanes 4,7) and wild-type cells (lanes 3,6) grown at permissive and restrictive temperatures, respectively (Sorger et al. 1995). The band-shift activity of a wild-type extract in the presence of an excess of wild-type (lane 2) or mutant (lane 1) CEN III DNA cold competitor is shown as a control, as well as the band-shift activity of a ndc10-1 extract from cells grown at 26°C (lane 5) or 37°C (lane 8). (B) Reconstitution of the microtubule-binding activity (number of beads bound per field) and the DNA-binding activity of a glc7-10 extract from cells grown at the permissive temperature by addition of baculovirus-produced Ndc10p, Cep3p, and Ctf13p–Skp1p. The reason for addition of Ctf13p–Skp1p as a complex rather than as separated proteins is explained elsewhere (Kaplan et al. 1997). The microtubule-binding activity has been normalized as described previously.