The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A
Figure 1
Scheme used for screening mutants having defects in microtubules or the nuclear envelope.
(A) For the screen, a strain was prepared that had the microtubule marker (GFP-Atb2), the nuclear envelope marker (Nup40-mCherry), and the spindle pole body marker (Sfi1-CFP) as well as the minichromosome. After chemical random mutagenesis, colonies were grown at 25°C and replica-plated onto two plates, which were then incubated at 36°C or 32°C. The plate at 36°C was used to examine temperature sensitivity, and the plate at 32°C (Low Ade) was used to identify colonies showing possible minichromosome loss. Colonies showing both temperature sensitivity and possible minichromosome loss were chosen for subsequent visual screening. Cell shape is outlined by dotted lines. Bottom: Colonies are shown schematically as white, black, or red circles. (B) Top: Schematic drawings for the centromeric region of chromosome III and for the minichromosome, CM3112sup3-5::ade6-M216-bsd, used in this study. CM3112sup3-5::ade6-M216-bsd contains a part of the centromeric region and the selection markers indicated. Bottom: The strain was streaked on nonselective media (N/S) and replica-plated onto both Low Ade and blasticidin S (BS) plates. Arrowheads indicate a colony that lost the minichromosome. (C) Cells with minichromosomes in the wild-type (WT) and the mis6-302 mutant background were streaked on Low Ade plates at 32°C for 5 (WT) or 8 days (mis6-302). (D) Summary of the screening results, showing the numbers of colonies obtained at each stage or category. (E) The population of mutants that showed unequal nuclear separation. Mutants were categorized based on whether they were dead or formed red or white colonies on Low Ade plates at 32°C.
