Figure 2.

rum1p is induced in G₁ in pheromone. (A) Western blot of extracts from P-factor–treated cells probed with anti-rum1p and control anti-α-tubulin antibodies. A lane with extracts from isogenic rum1Δ cells demonstrates that the slower migrating band cross-reacting with anti-rum1p is nonspecific. A cyr1Δsxa2Δ strain exposed to P-factor for 3 h at 25°C (left panel), cdc25–22cyr1Δsxa2Δ and cyr1Δsxa2Δ strains incubated at 36°C for 3 h and exposed to P-factor for 2 h at 36°C (middle panel), and a rum1Δcyr1Δsxa2Δ::REP6Xrum1⁺ integrant and a rum1⁺ control exposed to P-factor at 25°C for 4 h (right panel). (B) Cyr1Δsxa2Δ cells were nitrogen starved, and after 7.5 h, P-factor was added to half the culture. Extracts were Western blotted and probed with anti-rum1p and anti-α-tubulin antibodies. This anti-rum1p antibody shows no nonspecific cross-reacting band. (C) Northern blot of RNA samples from a cyr1Δsxa2Δ strain exposed to P-factor for 8 h at 29°C, probed for rum1⁺ and his3⁺ mRNA. (D) Protein extracts from ³⁵S-methionine pulse-labeled cyr1Δsxa2Δ cells before (C, −P) or after 3 h exposure to P-factor (+P), immunoprecipitated with preimmune serum (C), or anti-rum1p antibody (−P and +P). Because there was considerably more ³⁵S-methionine–labeled protein in the −P extract than in the +P extract, the rum1p-specific band was quantified and normalized with respect to an unspecific band. With this quantification, 120 relative units of ³⁵S were incorporated in the absence of pheromone, compared with 97 relative units in the presence of pheromone.