Identification of a FANCC-interacting protein by two-hybrid screening. (A) FANCC two-hybrid bait. The upper open rectangle represents wild-type FANCC. Asterisks indicate location of the R548X and L554P inactivating mutations.11,53,82 The lower open rectangle represents the region of FANCC expressed as a two-hybrid bait. The numbers indicate the corresponding amino acid residue of FANCC (the binding domain of the two-hybrid fusion is not shown for simplicity). The overlap with wild-type is indicated by the arrangement of the boxes in which the shaded area corresponds to the deletion in the IVS4 4A-T inactivating mutation that leads to a severe disease phenotype.14,53 (B) (see page 3740) Two-hybrid interaction. The Y190 yeast strain was cotransformed with the FAN bait plasmid and a representative prey plasmid (8-1) isolated by two-hybrid screening. Controls are shown for strains containing the prey plasmid 8-1 and an unrelated bait, and strains known to be positive and negative for interaction. The cotransformed strains are shown growing on media selective for both bait and prey plasmids (-Trp, -Leu), and under HIS selection. The lower panel shows results of the β-gal assay. (C) The protein encoded by clone 8-1 interacts with an amino terminal region of FANCC in vitro. Radiolabeled protein made by coupled in vitro transcription and translation from 8-1 cDNA bound specifically to immobilized GST-FANC1, but not to immobilized GST or GST-FAN2.