Figure 4

The flow chart used to screen the OHC library and steps for eliminating false positive clones. Yeast cells are transformed with bait plasmids containing the main gene of interest: Prestin, cdh23 or Alg5 (control bait) and with prey plasmids containing genes from the OHC library. If only one plasmid is transformed into the cell, the cell will die. If both prey and bait plasmids are transformed, but no interaction takes place between the resulting proteins, which would trigger the reconstitution of ubiquitin, the cell will live on double dropout plates but not on quadruple dropout plates. If prey and bait plasmids are transformed and there is an interaction between the resulting proteins, the cell will live on both double dropout and quadruple dropout plates. The colonies that grew on the quadruple dropout plates were then screened for false positives by replating on quadruple dropout plates containing X-gal, which turns blue in the presence of LacZ. Positive clones were screened by PCR. After prey plasmids were isolated from yeast and transformed into E. coli, they were purified and sequenced. Clones of interest were then retransformed into yeast cells along with the bait plasmid in order to confirm their interaction.