<i>gic1</i>Δ <i>gic2</i>Δ mutants spontaneously acquire a Mendelian suppressor mutation.

(A) gic1Δ gic2Δ mutants are inviable at 37°C. A diploid strain with the indicated genotype (DLY21711) was sporulated and tetrads (four spores in a vertical column) were dissected onto plates that were incubated at the indicated temperature. Tetrads contain two GIC1 gic2Δ spores and two gic1Δ gic2Δ spores. At 24°C all four spores were viable and gave rise to colonies, but at 37°C two spores from each tetrad died. Replica plating confirmed that the dead spores were the gic1Δ gic2Δ cells. (B) Isolation and genetic characterization of spontaneous gic1Δ gic2Δ suppressors. Cells of a gic1Δ gic2Δ strain (DLY20961) were streaked for single colonies. One million cells from each colony were plated on rich media and incubated at 37°C for 3 days. Although most cells died, several heterogeneously sized colonies were able to grow (example plate, bottom left), and one large colony from each independent plate was picked for further analysis. Suppressed cells were mated to a non-suppressed gic1Δ gic2Δ strain of opposite mating type (DLY21941), and the resulting diploids were sporulated and dissected as in (A). Tetrads showed 2:2 viability (middle panels) at 37°C indicating segregation of the suppressor as a single Mendelian locus. Independent suppressed strains (from different initial colonies) were then mated to each other and the resulting diploids were sporulated and dissected as in (A). All tetrads showed 4:0 viability at 37°C (right panels) indicating that the suppressors all map to the same locus. Sequencing confirmed that suppressed strains retained the gic1Δ and gic2Δ mutations.