Fig. 3. Mapping of the self-interaction domain. Truncations and deletions of the Hrp65 protein (or PSF in part B) were expressed as GAL4-AD or -BD fusions and assayed for their ability to interact with full-length Hrp65 in the yeast two-hybrid system. Graphical representations of the assayed constructs are shown on the left. (A) Mapping of the Hrp65 self-interaction domain. Yeast cells were transformed with a full-length Hrp65 construct plus a truncation/deletion construct. The protein-protein interactions were detected by plating serial dilutions of the co-transformants onto double selective -His -Ade medium. (B) ClustalW alignment of the protein binding domains (PBDs) of C. tentans Hrp65 (residues 259-415), Drosophila melanogaster NonA (residues 448-603) and Homo sapiens PSF (residues 443-601). Residues identical in all three sequences are shown in black boxes, identical residues in two out of three compared sequences are shown in dark grey and similar residues in two out of three compared sequences are shown in light grey. (C) Interaction of the PBDs of Hrp65 and PSF with the full-length Hrp65. Protein-protein interactions were detected as in (A), except that the transformants were selected on -His plates.

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