Figure 2. Germline Knockout of TCD1.

(a) Diagram of the TCD1 knockout. The neo3 drug resistance marker was inserted into the TCD1 gene, replacing most of the original coding sequence. (b) Southern hybridization of the TCD1 knockout strains. Total genomic DNA isolated from the knockout strains (ΔTCD1-6 and ΔTCD1-8) or WT cells was digested with Bgl II and hybridized with the TCD1 probe. Only the correct 3.4 kb band is observed for the knockout strains. (c) Viability of progeny. At 12 h to 13 h after mixing, single mating pairs were placed into drops of SPP medium and incubated for 48 h at 30 °C. (d) Division of progeny. ΔTCD1 cells were mated, and pairs were isolated in drops of SPP medium at 13 h to 14 h after mixing. The number of cells in each drop was counted before the cells died. The results were categorized as follows: 0, no cell division; 1, one division; 2, two cell divisions; 3, three cell divisions; 4, more than four cell divisions.