Yeast two-hybrid screening was performed by Hybrigenics Services SAS, Paris, France (www.hybrigenics-services.com). The coding sequence for Mus musculus–Wfs1 (amino acids 1 to 311; GenBank accession number GI:28422738) was PCR amplified and cloned into pB29 as a C-terminal fusion to LexA (N-Wfs1-LexA-C). The construct was checked by sequencing the entire insert and used as a bait to screen a random-primed mouse inner ear cDNA library constructed into pP6. pB29 and pP6 derive from the original pBTM116 (69, 70) and pGADGH (71) plasmids, respectively. Clones (54.3 million) (fivefold the complexity of the library) were screened using a mating approach with YHGX13 (Y187 ade2-101:loxP-kanMX-loxP, matα) and L40ΔGal4 (matα) yeast strains, as previously described (72). Twenty-nine His+ colonies were selected on a medium lacking tryptophan, leucine, and histidine. The prey fragments of the positive clones were amplified by PCR and sequenced at their 5′ and 3′ junctions. The resulting sequences were used to identify the corresponding interacting proteins in the GenBank database (National Center for Biotechnology Information) using a fully automated procedure. A confidence score [Predicted Biological Score (PBS)] was attributed to each interaction, as previously described (73).

The PBS relies on two different levels of analysis. First, a local score takes into account the redundancy and independency of prey fragments, as well as the distribution of reading frames and stop codons in overlapping fragments. Second, a global score takes into account the interactions found in all the screens performed at Hybrigenics using the same library. This global score represents the probability of an interaction being nonspecific. For practical purposes, the scores were divided into four categories, from A (highest confidence) to D (lowest confidence). A fifth category (E) specifically flags interactions involving highly connected prey domains previously found several times in screens performed on libraries derived from the same organism. Last, several of these highly connected domains have been confirmed as false positives of the technique and are now tagged as F. The PBS scores have been shown to positively correlate with the biological relevance of the interactions (74, 75).

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