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Fig. 3. The binding of both Mad and Zen to the Race enhancer is required
for proper Race expression. (A) DNAse I footprinting analysis of Mad
and Zen GST fusion proteins bound to a 255 bp fragment that includes the
proximal region of the Race 533 bp enhancer (349-533) and 70
nucleotides from the Bluescript vector. The fragment is end-labeled at the
vector end. Lane 1, chemical degradation of the probe on G+As; lanes 2 and 6,
DNAse I digestion of the DNA probe. Increasing amounts of Mad (500 ng, 1500 ng
and 4500 ng in lanes 3-5 respectively) and Zen (20 ng, 60 ng and 200 ng in
lanes 7-9 respectively) were incubated with the fragment prior to DNAse I
digestion. The region protected by Mad is depicted as a blue rectangle, the
hatched half denoting weaker protection. The regions protected by Zen are
shown as red ovals. The nucleotide sequence of the protected regions are shown
below the gel with the overlap between the Zen and Mad footprints shown in
purple. Putative core binding sites are underlined for Zen
(Han et al., 1989) and boxed
for Mad (boxes a,b,c,f,g) (Kim et al., 1996) and Medea (boxes d,e)
(Xu et al., 1998;
Pyrowolakis et al., 2004). The
boxes are also marked on the G+A sequence. (B) Schematic representation of the
full-length Race 533 bp enhancer fused to a lacZ reporter
gene, and a transgenic embryo carrying this construct in situ hybridized with
lacZ probes. lacZ expression is identical to the
Race pattern. The ring of staining in the head region is an artifact
of the vector. (C) Embryo carrying a deletion of the Mad-binding region
(nucleotides 432-497). lacZ expression is severely reduced. (D)
Embryo carrying mutations in the ATTA core sites of the Zen-binding sites
(underlined, see Materials and methods). lacZ expression is
absent.
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