Figure 6.

Both transactivation and Res2 binding domains are required for Rep2 function in fission yeast. (A) Rescue of low-temperature growth inability of rep2⁻ cells by expression of rep2⁺ deletion mutants. Full-length and various deletion mutants of rep2⁺ were inserted into the pcL vector and transfected into the rep2 disruptant (N3–141S) followed by selection at 18°C. The ratio of colonies formed at 18°C to those at 30°C is expressed as percentage suppression in the right column. pcL-X is the pcL vector with no insert and is used as a negative control. Experiments were repeated four times, and data are presented as means ± SD. (B) Flow cytometry of rep2 disruptants expressing each rep2⁺ deletion mutant. The rep2 disruptant was transfected with the indicated constructs and selected for Leu⁺ transformants. The transformants were grown to log phase at 30°C in PM medium and then incubated at 18°C for 53 h. Cells were sampled both at 30°C and after a 53 h incubation at 18°C, and analyzed by flow cytometry. (C) The level of cdc18⁺ mRNA expressed in the rep2 disruptants rescued by rep2⁺ deletion mutant genes. Total RNA was prepared from the rep2 disruptants at the same time points as described in B. The level of the cdc18⁺ transcript was determined by Northern blot hybridization. ura4⁺ was probed as a loading control. Lane 1, empty vector; lane 2, full-length rep2⁺; lane 3, Δ132–176 aa; lane 4, CΔ20; lane 5, Δ96–131 aa; lane 6, Δ22–95 aa; lane 7, ΔZn finger.