FIG. 7.

The ORF 50 carboxy-terminal mutation, 50M7, produces a stable protein which binds to the ORF 50 response elements. (a) Subcellular localization of ORF 50M7 protein, determined by immunofluorescence analysis of cells mock transfected (i) or transfected with pBK50M7 (ii). (b) OMK cells were seeded at 10⁶ cells per 35-mm-diameter petri dish and washed in labelling medium. Controls remained untransfected (lane 1) or transfected with 2 μg of pBK50b (lane 2) or pBK50M7 (lane 3). The cells were incubated in labelling medium, harvested, and then lysed after 24 h. For each immunoprecipitation, 20 μl of the anti-ORF 50 polyclonal antibody was incubated with protein A-Sepharose beads for 16 h at 4°C. Immunoprecipitations were then performed with each cell lysate, using the anti-ORF 50 antibody. These samples were then pelleted, resuspended in Laemmli buffer, resolved on an SDS–12% polyacrylamide gel, and analyzed by autoradiography. (c) Gel retardation assays were performed as previously described (53). Briefly, the ORF 50 response elements contained in a set of oligonucleotides were annealed, radiolabelled and then incubated with nuclear extracts of untransfected OMK cells (lane 1) or cells transfected pBK50b (lane 2) and pBK50M7 (lane 3). The protein-nucleic acid complexes (indicated by the arrow) were separated on a 5% polyacrylamide gel, run in 1% TBE buffer, and detected by autoradiography.