FIG. 3.

Schematic representation of the IglA in-frame deletions (A) and point mutants (B) fused to the GAL4 activation domain of plasmid pGADT7. All constructs were cotransformed with IglB in the GAL4 DNA-binding domain plasmid pGBKT7 into the S. cerevisiae two-hybrid system reporter strain AH109. The ability of each mutant to bind IglB was recorded as the degree of activation of two independent reporter genes, ADE2 and HIS3, that permitted growth of the yeast on minimal medium devoid of adenine and histidine, respectively, after day 5 with incubation at 30°C, which was expressed using a scale from ++++ (wild-type growth) to − (no growth). The results reflect the trends in growth based on three independent experiments in which several individual transformants were tested on each occasion. To measure activation of the lacZ reporter, constructs were introduced into the S. cerevisiae reporter strain Y187. The mean ± standard deviation β-galactosidase (β-gal) activities produced by mutants compared to wild-type IglA in two independent experiments in which several transformants were tested on each occasion are indicated. In panel A the relative positions in the full-length IglA construct of the four α-helices, H1 (amino acids 62 to 68), H2 (amino acids 102 to 128), H3 (amino acids 133 to 143), and H4 (amino acids 146 to 157), are indicated. In panel B the amino acid sequence for residues 102 to 128 predicted to form α-helix H2 is shown. Amino acids that were replaced with alanine are indicated by filled triangles.