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Fig. 4. Identification of multiple positive-acting elements within the M250
enhancer for motoneuron expression. (A) Point mutations were introduced into
nine sites (M1-M9) as indicated. (B-K) GFP expression in HH stage 24 chick
embryos with each mutant reporter tested in the context of the
M250+
NheI:GFP construct (see
Fig. 1B,F). (L) E11.5
transgenic mouse embryo generated with the M250 reporter containing M1 and M9
mutations and stained with anti-GFP antibody. (M) Location of oligonucleotides
(indicated by rectangles) in the Hb9 promoter used for gel
retardation assays. The sequence of these regions is highly conserved between
mouse and human. (N) The binding of Isl1/Lhx3 to the M50 DNA segment was
challenged by 20-, 100-fold molar excess of the unlabeled probes indicated
above the lanes. M50 and M100C competed efficiently for Isl1/Lhx3 DNA binding,
while the M3-oligo and 3'CD-oligos failed to do so. This suggests the M3
element is not a binding site for the LIM-HD factors Isl1 and Lhx3.
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