Fig. 4. Mrl-binding sites in the tll promoter. (A) The binding of Mrl to sites 1 and 2 was assayed by a gel mobility shift assay using synthetic oligonucleotides corresponding to the two sites and surrounding sequences. The Hop/Mrl pathway was activated by treating S2 cells with vanadate-H₂O₂ (Sweitzer et al., 1995). We find that site 1 binds strongly to Mrl, while the affinity of site 2 is lower. Addition of anti-Mrl antibody produced a supershift for each protein-oligo complex, which is consistent with the binding of Mrl to these sequences. (B) The positions of the two Mrl-binding sites are shown relative to the tll transcription start, and their sequences are shown and compared with STAT-binding consensus and optimal Mrl-binding sequences. (C) The 5.9 kb regulatory fragment upstream from the tll transcription start site is sufficient to drive lacZ expression in a pattern similar to that of endogenous tll in wild-type embryos. (D) In tor^GOF embryos, this promoter fragment drives lacZ expression in expanded domains. (E) A mutant 5.9 kb fragment was generated by disrupting both Mrl-binding sites (see Materials and Methods). In wild-type embryos, the expression pattern of lacZ driven by this mutated 5.9 kb fragment was not affected. (F) However, in tor^GOF embryos, the expansion of lacZ expression pattern was reduced.

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