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Fig. 4. MOG-6 binds to MEP-1. (A) Interaction assays between MOG-6 and additional regulators of fem-3 in the yeast two-hybrid system. Growth on medium lacking histidine (left) or blue staining (right) represents positive interaction. MOG-6 was fused to the LexA DNA-binding domain, while the other proteins were fused to the Gal4 activation domain. Bottom lane, MOG-6 does not bind to itself. (B) Binding of MOG-6 to MEP-1 in vitro. MOG-6 bound to GST-MEP-1(FL) or GST-MEP-1(SH), but neither to GST alone nor to GST-FBF-1. (C) Functional domains in MEP-1. MEP-1(FL) is represented schematically in row 1: the Q-rich domain is shown as a blue box, and the zinc fingers are represented by vertical triangles. Row 2, MEP-1(SH) lacks the N-terminus (residues 1-407). Rows 3 to 22 represent other deletion derivatives of MEP-1. The shortest MEP-1 derivative that interacted with MOG-6 comprised 111 residues of the N-terminus (row 17, red box, residues 123 to 233) or 361 residues of the C-terminus (row 20, red box, AA 510 to 870). Internal deletions are shown by bars joining the N- and C-termini (rows 5 to 8). (D) mog-6 rescue and MEP-1 binding. The complete mog-6 gene and derivatives thereof were tested for mog-6 rescue. The wild-type mog-6 gene rescued mog-6 (row 1, 9% rescue, n=31 rolling unc-4mog-6 worms analyzed; self-fertile rolling Uncs were considered as rescued). mog-6 constructions bearing point mutations in the CBD (bars in rows 2 to 4) or lacking the entire CBD (AA275-434; row 5) rescued mog-6 at least to the same extent as the wild-type mog-6 gene. MOG-6 truncations retaining either the entire CBD (row 6), the CBD plus the C-terminus (row 7), or the C-terminus alone (row 8) did not rescue mog-6. Similarly, the entire N-terminal extension of MOG-6 did not rescue mog-6 (row 9). MOG-6 derivatives were also assayed for MEP-1 binding in the yeast two-hybrid system. Right; MOG-6 requirements for MEP-1(FL) binding. Full length MOG-6 bound to MEP-1 (row 1, see also Fig. 4A). A MOG-6 protein that lacked the CBD interacted with MEP-1 (row 5), but neither the CBD alone (row 6), nor the CBD fused to the C-terminus (row 7) were sufficient for MEP-1 binding. If taken separately, the C- and N-termini of MOG-6 did not interact with MEP-1 (rows 8 and 9).





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