Figure 4.

Effects of orb6 overexpression. (a–c) Overexpression of HA-tagged orb6⁺ in a leu1–32 strain (experiment 1 in Table 1). (a) leu1–23 pRep3X. (b) leu1–23 cells, carrying integrated HA-tagged orb6⁺, were grown exponentially for at least eight generations at 32°C in the absence of thiamine. (c) leu1–32 cells, carrying HA-tagged orb6⁺ on the multicopy plasmid pRep3X, were grown in the absence of thiamine for 18 hr at 32°C. (d–f) Overexpression of HA-tagged orb6⁺ in a wee1–50 mutant strain. Cells were grown exponentially at 25°C in the presence of thiamine for at least eight generations; thiamine was washed away and cells were grown in the absence of thiamine at 25°C for 16 hr, then shifted at 36°C for 5 hr. (d) leu1–32, pRep3X orb6HA. (e) wee1–50 leu1–32 pRep3X. (f) wee1–50 leu1–32 pRep3X orb6HA. (g) Western blot comparing the levels of expression of orb6-HA in the experiments described above, as compared with the control tubulin: lane 1, leu1–32 with an integrated copy of orb6-HA; lane 2, leu1–23 pRep3X; lane3, leu1–32 pRep3x orb6 HA; lane 4, leu1–32 pRep3x orb6 K122A HA; lane 5, wee1–50 leu1–32 pRep3x; lane 6, wee1–50 leu1–32 pRep3x orb6-HA. (Bar = 5 μm.)