Figure 7.

Synchronized depleted SSU processome proteins arrest in G1 upon cell cycle release. GAL::RPS14A, GAL::UTP1, GAL::UTP2, GAL::UTP4 yeast strains bearing Δbar1, Cln2-TAP, Clb2-3xHA were grown to early log phase in galactose/raffinose media (undepleted) and then shifted to glucose (depleted) for 21 h. a-Factor (2 μg/ml) was added to cells for 2.5 h and then washed out of the media. (A) Western blot on synchronized and released yeast. Protein was extracted from equal amounts of cells every fifteen minutes after the α-factor release and Western blotted with PAP antibodies (to visualize Cln2-TAP) and anti-HA antibodies (to visualize Clb2-3xHA). (B) FACS analysis of synchronized and released yeast. Cells were arrested with α-factor for 2.5 h and analyzed for DNA content (1C, 1 DNA content; 2C, 2 DNA content) by FACS. After 105 min of release from α-factor, yeast were again analyzed for DNA content by FACS sorting.