Fig. 4. Two-hybrid interactions between Ten1, Stn1 and Cdc13. (A) Ten1 associates with Cdc13, as well as with Stn1, in a yeast two-hybrid system. Strains simultaneously expressing pAS2-TEN1 and pACT2-STN1 (bottom row, Ten1 + Stn1) or pAS2-TEN1 and pACT2-CDC13 (bottom row, Ten1 + Cdc13) were positive for β-galactosidase activity, measured as described in Materials and methods, like cells co-expressing pAS2-CDC13 and pACT2-STN1 (bottom row, Cdc13 + Stn1) used here as a positive control. On the other hand, cells simultaneously expressing pAS2-TEN1 and pACT2 alone (top row, Ten1), pAS2 alone and pACT2-CDC13 (top row, Cdc13) or pAS2 alone and pACT2-STN1 (top row, Stn1) scored negative in the X-gal assay. Patches of cells replica-plated on nitrocellulose membrane were incubated for 2 h at 30°C and then photographed. For each one of the six pAS2/pACT2 pairs shown here, three different strains were monitored (four transformants for each). (B) The Stn1-13 mutant protein fails to interact with either wild-type Ten1 or wild-type Cdc13 at restrictive temperature. The stn1-13 allele was expressed from pACT2 together with pAS2-TEN1 or pAS2-CDC13, as explained above. Both pairs scored negative for β-galactosidase activity (white patches) at 34°C, but not at 25°C (permissive temperature), while the corresponding controls expressing wild-type STN1 were positive at both temperatures (blue patches). (C) The Ten1-6 mutant protein still interacts with both wild-type Stn1 and wild-type Cdc13. The ten1-6 allele was expressed from pAS2 together with pACT2-STN1 or pACT2-CDC13, as explained in (A). Both pairs scored positive in the X-gal assay (blue patches) at 30°C (ten1-6 is not a ts allele), similarly to the corresponding controls expressing wild-type TEN1.