Figure 5.

Pas1 cyclin negatively regulates sexual differentiation. (A) Mating efficiencies of homothallic wild-type (L968) (open symbols) and Δpas1 mutant (K207-A1) (closed symbols) cells in nitrogen-rich MM. Cells were grown to log phase in MM (+N/2%G) and transferred to MM (+N/2%G) (circles) or MM (+N/0.5%G) (squares), and mating frequencies were assayed. (B) Mating efficiencies of homothallic wild-type (open symbols) and Δpas1 mutant (closed symbols) cells in MM without nitrogen. Cells were grown to log phase in MM (+N/2%G) and transferred to MM (−N/2%G) (circles) or MM (−N/0.5%G) (squares), and mating frequencies were assayed. (C) sxa2⁺ transcription is induced without nitrogen starvation in Δpas1 cells by P-factor. h⁻ wild-type (L972) and h⁻ Δpas1 (K193-A1) cells were inoculated into MM (+N/0.5%G) and cultured in the presence (+) or absence (−) of 2 μg/ml P-factor. Cells were harvested at the indicated times, and sxa2⁺ expression was examined by Northern hybridization. (D) Δpas1 cells are hypersensitive to mating pheromone and arrest the cell cycle without accompanying nutrient starvation. h⁻ Δsxa2 (K205-A1) (referred to as wild-type; open symbols) and h⁻ Δsxa2 Δpas1 (K209-22A) (referred to as Δpas1; closed symbols) cells were inoculated into MM (+N/0.1%G), divided into two parts, incubated at 30°C for 4.5 h, then cultured in the presence (+; circles) or absence (−; squares) of 2 μg/ml P-factor. Cell growth was monitored by counting cell numbers. (E) Δpas1 cells arrest in G₁ phase upon exposure to P-factor. The same cell cultures used in D were harvested at 4 h after P-factor addition and analyzed by flow cytometry. (F) Δrep2 cells mate as efficiently as Δpas1 cells. Homothallic wild-type (L968), Δpas1 (K207-A1), Δrep2 (K166-A1), and Δnrd1 (14-1) cells were grown to log phase in MM (+N/2%G), transferred into MM (+N/0.5%G) or MM (−N/2%G), and cultured at 30°C. Mating efficiencies were determined by examining mated cells after 24 h of incubation. (G) Deletion of pas1⁺is not epistatic to deletion of nrd1⁺. Homothallic wild-type (L968), Δpas1 (K207-A1), Δnrd1 (14-1), and Δnrd1 Δpas1 (K228-29A) cells were grown to log phase in MM (+N/2%G) and transferred into MM (+N/0.5%G). Mating frequencies were determined as described above.