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. 2000 Apr;11(4):1225–1239. doi: 10.1091/mbc.11.4.1225

Figure 1.

Figure 1

Two mutations genetically link Pkl1p and γ-tubulin. (A) Synthetic lethality strategy with strain genotype (see MATERIALS AND METHODS for details). The pkl1 gene is deleted in the genome (bars represent three chromosomes; dark is the pkl1 locus) and replaced by a plasmid (circle) carrying wild-type pkl1+ and ade1+. Red and white screening uses ade1 and ade6 mutations (Moser et al., 1995). Ade6− cells appear red on low-adenine plates, whereas [ade1−, ade6−] cells are white. Mutations that cause plasmid dependence result in red cells, because ade1− is complemented. S. cerevisiae LEU2+ complements S. pombe leu1− and is a genetic marker in lieu of ade1+ on control and test plasmids. Control plasmids: (negative) pREP [LEU+, no pkl1]; (positive) pSMpkl1 [LEU+, pkl1+]. (B) Cold-sensitive growth of SL1 and SL2 synthetic lethal mutants at 19°C compared with wild-type S. pombe 972 h−. (C) Photograph of condensed chromosomes in SL1 and SL2 cells at 19°C, stained with Hoechst. Microtubule and Hoechst combined images acquired by CCD camera are shown for SL1 only in Figure 2. The bottom image is an enlargement of chromosomes in SL1. Bar, 5 μm. SL1 cells arrest in mitosis and only rarely proceed to cytokinesis. (Arrows in the upper panel point to a septum in two cells. DNA is off center.) (D) SL1 and SL2 cells, transformed with plasmid controls (pREP and pSMpkl1) and tubulin genes (see this section of RESULTS), were streaked onto selective plates (EMM, no leucine) containing low adenine. Duplicate transformants for each tubulin gene are shown. Red indicates that the original plasmid [pkl1+, ade1+, ars1] is still present and required for growth. White cells have lost the original plasmid. Labels for streaks in SL2 are as shown for SL1. (E) Disruption of the checkpoint gene mad2 in SL1 cells with single-copy pkl1 removes the cold-sensitive mitotic block. (F) Tetrads tested for growth at 19°C. SL1 with single-copy pkl1 was crossed to strain h+ nda2-340, which carries a cold-sensitive microtubule-destabilizing mutation in α-tubulin (see last section of RESULTS). Only the parental genotypes remained cold sensitive.