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Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos.
Bell P
,
Mais C
,
McStay B
,
Scheer U
.
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When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.
Fig. 1. Immunofluorescence microscopy of Xenopus XTC-2 cells with antibodies directed against xUBF (A¢), xTBP (B¢) and pol I (C¢). All
three antibodies stain the nucleoli. An additional labelling of the nucleoplasm at various intensities can be observed with the antibodies to xTBP
(B¢). The corresponding phase contrast images are shown in A-C. Bar, 20 um.
Fig. 2. Immunofluorescence microscopy
of nuclei assembled from sperm
chromatin in Xenopus egg extract with
antibodies directed against xUBF.
Besides being diffusely distributed
throughout the nuclear interior, UBF is
highly concentrated at one specific site
per nucleus. These UBF-foci appear
either as brightly fluorescent dots (A¢) or
necklace-like structures consisting of
tandemly arrayed fluorescent granules
(B¢). The corresponding DNA-staining
with Hoechst dye 33258 is shown in A
and B. Bar, 10 um.
Fig. 3. The TATA-box binding protein (TBP) is not concentrated at specific sites
but rather dispersed throughout the interior of the in vitro assembled nuclei (A¢¢).
The corresponding phase contrast image (A) and Hoechst fluorescence (A¢) are
shown. Bar, 10 um.
Fig. 4. UBF, pol I, and fibrillarin localize at three different sites within nuclei assembled in Xenopus egg extract. (A) Double label
immunofluorescence microscopy with antibodies directed to pol I (A¢) and UBF (A¢¢) reveals that pol I is concentrated at several sites scattered
throughout the nucleoplasm (A¢) but not at the UBF-positive spot (A¢¢). (B) Double label experiment with antibodies against pol I (B¢) and
fibrillarin (fib; B¢¢). The pol I foci (B¢) are not identical with the nuclear bodies which contain a specific set of nucleolar proteins such as
fibrillarin (B¢¢). The corresponding Hoechst fluorescence is shown in A and B. Bar, 10 um.
Fig. 5. Nuclei assembled from sperm chromatin in UBF-deficient egg extract lack UBF-positive structures. Egg extracts were incubated with
paramagnetic beads coated with either UBF antibodies or the corresponding preimmune IgGs. After retrieval of the beads, samples of the
extracts and proteins bound to the beads were analyzed by immunoblotting experiments using the same antibodies. (A) UBF is detectable in
control extract after incubation with preimmune IgGs (lane 1, arrow) but not in extract treated with UBF-antibodies (lane 3). Additional protein
bands of 130 and 200 kDa are also stained by the UBF-antibodies on the western blot (lane 1; note, that these two proteins are not removed
from the extract along with UBF, see lane 3). UBF is recovered from the beads with UBF-antibodies (lane 4, arrow) in contrast to the control
beads (lane 2). The bands marked H in lanes 2 and 4 represent the heavy chain of the antibodies bound to the beads. As a control, nuclei were
assembled in extract treated with preimmune IgGs. After immunostaining with UBF-antibodies these nuclei display characteristic UBF-foci
(B¢). Nuclei assembled in UBF-deficient extract are completely negative with UBF antibodies (C¢). The corresponding Hoechst fluorescence is
shown in B and C. Bar, 10 um.
Fig. 6. Nuclei reconstituted from l-DNA in egg extract accumulate
UBF as shown by immunofluorescence with UBF-antibodies (A¢).
However, they lack the UBF-foci. DNA staining with Hoechst is
shown in A. Bar, 20 um.
Fig. 7. Nuclei assembled
from sperm chromatin of
wild type (A, A¢) and 1-nu
Xenopus mutants (B, B¢)
analyzed by
immunofluorescence with
UBF-antibodies. Almost all
nuclei derived from sperm
chromatin of wild-type
(2-nu) animals reveal the
characteristic UBF-focus
(A¢). In contrast, about half
of the nuclei assembled from
sperm chromatin of 1-nu
mutants lack the UBFpositive
dot (B¢). The
corresponding Hoechst
fluorescence is shown in A
and B. Bars, 10 um.
Fig. 8. UBF colocalizes with the rRNA genes. Nuclei assembled from sperm chromatin were first stained with antibodies to UBF (A) and then
subjected to in situ hybridization with a digoxigenin labelled probe complementary to part of the 28S region of Xenopus rDNA (A¢). Both
procedures label the same dot-like structure. The corresponding Hoechst fluorescence is shown in A¢¢. The slightly different contours of the
nuclei is a result of the hybridization procedure. Bar, 10 um.
Fig. 9. Immunofluorescence microscopy of squash
preparations of Xenopus embryos before (A¢,B¢) and
after (C¢) the midblastula transition following
incubation with antibodies to UBF. A dividing
blastomere of a 64 cell-stage embryo in metaphase
reveals two UBF-positive dots (arrows in A¢).
Likewise, nuclei of an early blastula also contain two
UBF-dots each (indicated by arrows; the chromatin
of the two cells shown is heavily distorted due to the
spreading procedure). After midblastula, the
embryos eventually develop nucleoli which can be
readily stained with UBF-antibodies as exemplified
in a tailbud stage embryo (C¢). The corresponding
Hoechst fluorescence is shown in A-C. Bars, 20 um.