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Fig. 5. Evidence for DNA damage in Arabidopsis csn mutants.
(A) Accumulation of the phosphorylated H2AX variant 
H2AX in a
histone preparation (13 µg) from 2-day-old (same size) and 7-day-old (same
age) wild-type seedlings and 7-day-old csn3, csn4 and csn5ab
mutant seedlings as detected with a H2AX-specific antibody
(Friesner et al., 2005). A
Coomassie-stained band serves as a loading control. H2AX
transcription was not altered between the wild type and the csn
mutants (data not shown). (B,C) Immunostaining of wild-type and
csn4 mutant root tip cells with the anti-H2AX antibody (green)
identifies subnuclear H2AX-specific foci that may mark sites of damaged
DNA in csn4 mutants (C, left panel). The samples were counterstained
using the DNA stain DAPI. Scale bars: 5 µm. (D) Activation of the
stress-induced WEE1:GUS reporter construct in wild-type seedlings,
bleomycin-treated (12 hours, 5 µg/ml) wild-type seedlings and in untreated
csn mutants. Scale bars: 2 mm in wild type; 0.5 mm in csn3,
csn4 and csn5ab. (E-M) Confocal images of primary roots
of 7-day-old dark-grown wild type and csn mutants following the TUNEL
assay (left column). Roots were counterstained with DAPI (right column). For
the positive control, fixed root material was subjected to treatment with
DNase I. For the negative control, terminal transferase was omitted from the
TUNEL reaction. The experiment was repeated three times and a representative
image from one experiment is shown. Scale bars: 1 mm in wild type,
csn5a and csn5b; 0.5 mm in csn3, csn4 and
csn5ab. (N) Confocal images of a 7-day-old bleomycin-treated
(12 hours, 5 µg/ml) dark-grown wild-type seedling following the TUNEL assay
(left panel). Roots were counterstained with DAPI (right panel). Scale bar: 1
mm.
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