Fig. 3. Regulation of SG2 by Fkh is direct. (A) 885 nucleotides of sequence upstream and spanning the translation start site of SG2 (green letters) was sufficient to direct Fkh-dependent salivary gland expression of the lacZ reporter gene. Although the 885 nucleotide sequence contains four putative Fkh-binding sites (A, blue ovals and blue sequences), only three of the sites (f,g,h, blue filled ovals) were bound by Fkh protein in vitro or competed for binding (Aa, Ab). Whereas both unlabelled wild-type h and wild-type f compete well for binding with P³²-labelled wild-type h, neither mut h nor mut f compete for binding (compare left to right gels in Aa and Ab). Adding cold competitor in all cases reduced the amount of labeled oligo that failed to enter the gel, making it appear as if the addition of mutant oligos increased the specific binding of Fkh to the labeled oligo, especially on the gel on the right with the mut site f competitor. Six consensus bHLH binding sites (CAXXTG) are present in the 885 nucleotide SG2 enhancer (A, red circles and red sequences). (B-D) Embryos carrying a wild-type SG2 885-lacZ construct had ßgal expression in the salivary glands (arrows) beginning at stage 11 and continuing through embryogenesis. (E-G) Embryos carrying the SG2 885 fkh-lacZ constructs with mutations in the three Fkh in vitro binding sites (f,g,h) had reduced salivary gland expression at early stages (arrow indicates the area outlined in black, which is the unstained salivary gland) and variable expression at later stages (arrows in F,G). (H,I) The salivary gland expression (arrows) of the SG2 885-lacZ construct visible in wild type (H) was absent in the salivary glands of fkh mutants (I). Embryos in H,I were stained in the presence of NiCl.