Figure 3.

chl1 cells are proficient in kinetochore-microtubule interactions. A. Wild-type and chl1 mutant transformants carrying the dicentric plasmid show similar colony morphology. AP22 (wild-type), AP22Dchl1 (chl1) and M29/5D (ctf19/mcm18-1) cells were transformed with a centromeric plasmid YCp50 and its dicentric derivative YCp50-5 and plated on selection plates. The plates were incubated at 30°C for 3 days. SC-URA refers to synthetic complete medium lacking uracil. B. Panel 1. YCp50-5 was recovered from 9 transformants from each of wild-type and chl1 mutant cells. Recovered DNA, digested with PstI, was fractionated by electrophoresis on a 0.7% agarose gel. Lanes 1-9 and Lanes 11-19 show recovered YCp50-5 from wild-type and mutant cells respectively. Lane C is control DNA of YCp50-5, isolated from E. coli, digested with PstI. Lane M shows λ-DNA digested with HindIII. Panel 2. YCp50-5 recovered from ctf19 transformants does not show breakage of DNA. YCp50-5 DNA was recovered from seven M29/5D (ctf19/mcm18-1) transformants, digested and fractionated as described in Panel 1. Lane M contains λ-DNA digested with HindIII C. Kinetochores of chl1 cells show bipolar connections in the presence of HU. Exponentially growing US3329 (wild-type) and US3329Δchl1 (chl1) cells were treated with 0.2 M HU for 4 hours. Elongated mitotic spindles having separated CEN5-GFP dots (yellow dots in the merged field) in chl1 cells indicates bipolar connections of kinetochores.