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Fig. 2. Ectopic tethering of lacI-JIL-1 fusion protein to a polytene band
induces histone H3S10 phosphorylation and chromatin decondensation.
(A-C) Triple labelings of polytene squash preparations from third
instar Drosophila larvae homozygous for the lacO repeat line
P11.3, which is inserted into the middle of a polytene band in region 96C1-2.
GFP-LacI was tethered to the lacO repeats in A and LacI-JIL-1 in B,C.
GFP-, LacI- and JIL-1-antibody labeling is shown in green, H3S10ph-antibody
labeling in red and Hoechst 33258 labeling of DNA in blue or gray. The white
arrows indicate the lacO repeat insertion site. The polytene
chromosomes from the three preparations are aligned to show the `split' in the
polytene bands, reflecting decondensation of the chromatin when lacI-JIL-1
fusion protein is tethered to the band, in contrast to its wild-type
morphology, when GFP-LacI is tethered and there is no ectopic upregulation of
histone H3S10 phosphorylation. Note: the endogenous JIL-1 and H3S10ph antibody
labeling is too weak relative to the LacI-JIL-1 signal and the induced
hyperphosphorylation of H3S10 to be clearly visible at this exposure level.
(D) Without a GAL4-driver line there is no LacI expression or
changes to the band/interband structure. Double labelings with LacI antibody
(in green) and Hoechst 33258 (in blue or gray) of polytene squash preparations
from third instar larvae homozygous for the lacO repeat line P11.3
and containing a LacI-JIL-1 transgene but without a GAL-4 driver.
Arrows indicate the approximate lacO repeat insertion sites.
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