|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 4. The site of crucial importance for in vivo activity of enhancer
EVIII is specifically recognized by Lab and its two co-factors Exd
and Hth. (A) Band-shift experiments showing that Lab (lane 3), Lab/Exd (lane
4) and Lab/Exd/Hth (lane 5) specifically bind to the oligonucleotide
EVIII. Specificity of the binding was verified by adding a polyclonal
anti-Lab antibody to the reactions (lanes 6-8), or by mutating in a G stretch
the putative core binding sites of Lab (lanes 11-13), Exd (lanes 16-18) and
Hth (not shown). We noted that the Exd-binding site mutation affected Lab
monomere binding in presence of the co-factor (lanes 17-18). No different
DNA-binding activities were observed between the truncated (158-629) and the
full-length form of Lab on the consensus Lab/Exd/Hth-binding site of the
lab48/95 enhancer (lanes 19-26). Lanes 1, 9, 14 and 19 are probes
alone. Lanes 2, 10, 15 and 20 are probes in presence of the rabbit
reticulocyte lysate. For each gel, the following lanes correspond to the Lab
protein alone (3, 6, 11, 16, 21 and 24), with Exd (4, 7, 12, 17, 22 and 25),
or with Exd and Hth proteins (5, 8, 13, 18, 23 and 26). Lab proteins were full
length, except in lanes 21-23, where the 158-629 form (see Materials and
methods) described in previous studies was used. (B) Gel mobility experiments
with oligonucleotide EVIII, in presence of the Hox proteins Lab
(lanes 3-5), Proboscipedia (Pb; lanes 6-8), Sex combs reduced (Scr; lanes
9-11), Deformed (Dfd; lanes 12-14), Antennapedia (Antp; lanes 15-17),
Ultrabithorax (Ubx; lanes 18-20), AbdominalA (AbdA; lanes 21-23) and
AbdominalB (AbdB; lanes 24-26). Hox proteins are alone (lanes 3, 6, 9, 12, 15,
18, 21, 24), with Exd (4, 7, 10, 13, 16, 19, 22, 25) or with Exd and Hth (5,
8, 11, 14, 17, 20, 23, 26). Exd and Hth proteins correspond to the truncated
isoforms described in previous studies (see Materials and methods). Lanes 1
and 2 correspond to probe alone or with rabbit reticulocyte lysate,
respectively. Hox/Exd or Hox/Exd/Hth complexes are only observed with Lab
(lanes 4 and 5). Lab proteins were full length, except in lanes 3-5. Lab
binding, alone or in combination with the Exd and Hth co-factors, is indicated
by arrowheads.
|
