Fig. 4. Binding of SEM-4 to the mec-3 and egl-5 promoters. (A) Binding to the mec-3 promoter. The indicated changes were made to inactivate the sites in the specific competitor oligonucleotides for the gel shift. The addition of SEM-4 to radiolabeled probe produced four complexes (arrows). Binding decreased substantially in the presence of 100-fold and 50-fold molar excess of cold specific competitor. Binding did not decrease as much when mutated competitor was added. Mutation of m3-1, from AGACAA to AGCTAG, restored some of the binding; mutation of both m3-1 (to AGCTAG) and m3-2 (from ACACAA to ACCTAG), restored more of the binding. The sequence of m3-3 is ACACAA. (B) Binding to a region of the egl-5 promoter close to the translation start site. Control protein was prepared from cells transformed with the pGEX6P-1 vector, lacking the sem-4 cDNA insert. The addition of SEM-4 to radiolabeled probe produced three complexes (arrows). Binding decreased substantially in the presence of 100-fold molar excess of cold specific competitor. Mutation of e5-1 (from TTGTGT to CTAGGT), e5-2 (from TTGTCT to CTAGCT) and e5-3 (from ACACAA to ACCTAG), in the specific competitor restored binding of complexes 1 and 3. (C) Ectopic T lineage expression of PV6CREgfp in sem-4 animals and of PV6CRE100gfp in wild-type animals. PV6CREgfp in wild-type animals shows only occasional, faint T lineage expression (top). Scale bar: 10 µm. Arrows indicate T.pa and T.pp cells expressing GFP. (D) Binding to V6CRE. The addition of SEM-4 to radiolabeled probe produced two complexes (arrows). Binding decreased substantially in the presence of 100-fold molar excess of cold specific competitor. Mutation of e5-4 and e5-5 as indicated in the specific competitor restored binding of complexes 1 and 3. Mutation of e5-T1 as indicated did not restore binding. Mutation of e5-4, e5-5 and e5-T1 produced the same restoration of binding as mutation of e5-4 and e5-5.

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