Fig. 4. Effect of over-expressing Pmt3 and Pmt3-GG in wild-type and ulp1.d cells. (A,B) Total cell extracts from wild-type (sp.011) (lanes 1-3) and ulp1.d (sp.651) cells (lanes 4-6) transformed with pREP41HA (lanes 1,4), pREP41HA-Pmt3 (lanes 2,5) or pREP41HA-Pmt3-GG (lanes 3,6) and grown for 18 hours in selective medium lacking thiamine were analysed by western blotting with anti-Pmt3 antisera (A) or anti-HA antisera (B). Equal amounts of protein were loaded in each lane as determined by staining with Coomassie Brilliant Blue (data not shown). (a) Pmt3-GG, (b) full-length Pmt3, (c) HA-Pmt3-GG, (d) HA-Pmt3.

(C,D) Wild-type (sp.011) and ulp1.d (sp.651) cells transformed with pREP41HA, pREP41HA-Pmt3 or pREP41HA-Pmt3-GG were grown for 12 hours to midlog phase in selective medium lacking thiamine. Cells were then diluted (t=0) to 10⁶ cells/ml in fresh thiamine-free medium and counted every 2 hours.

(E) Transformed cells were grown for 18 hours to 4x10⁶ cells/ml in selective media lacking thiamine and then subjected to UV radiation as indicated.