FIGURE 3:
Overexpression of Tti1, but not Tel2 or Tti2, allows growth of sis1∆ cells. (A) Tenfold serial dilutions of cells having indicated TTI1 and SIS1 on the chromosome (chrom) or expressed from a plasmid were plated on rich media. Overexpression from the ADH1 promoter indicated by an upward arrow. Plates were incubated for 3 d at indicated temperatures, except 23°C plates for 5 d. (B) Lysates of sis1∆ cells having HA-tagged TEL2 and Flag-tagged TTI2 in the chromosome and indicated proteins expressed from centromeric plasmids were subjected to electrophoresis and immunoblot analysis using antibodies against HA and Flag tags. Sis1 under control of TET promoter (TET-SIS1) with doxycycline added 21 h before harvest (+) or, as a control, no addition (–); suppressor Ydj1G70N from its native promoter; HA-tagged TTI1 or tti1sup#1 (tti1S#1) from the strong ADH1 promoter (upward arrow). (C) Transformants of sis1∆ strain carrying SIS1 on a URA3-based plasmid expressing the indicated protein or vector control (–) were streaked onto plates containing 5-FOA and incubated for 4 d at 30°C.