FIGURE 14.

Cells expressing sld2-K44E,K50E,K438E exhibit a severe growth defect with very slow progression into S phase. A, 10-fold serial dilutions of sld2-td cells expressing SLD2 or sld2-K44E,K50E,K438E at the restrictive (37 °C) or permissive temperature (24 °C). B, Western blot analysis of SLD2 and sld2-K44E,K50E,K438E whole cell extracts showing equivalent levels of Sld2 expression under restrictive conditions. C, sld2-td cells expressing SLD2 or sld2-K44E,K50E,K438E cultured in YP medium with 2% raffinose (YPRaff) to logarithmic phase at 24 °C and synchronized at G₁ phase by incubation with α-factor. Cells were cultured first in YPRaff for 1 h at 24 °C then in YP medium with 2% galactose (YPGal) for 1 h at 24 °C, and finally in the same medium for 2 h at 37 °C in the presence of α-factor. Cells were washed and released into the fresh YPGal and cultured at 37 °C. Upper, experimental outline and timing of sampling. Lower, FACS analysis.