Heat shock induces mini-Cajal bodies in the Xenopus germinal vesicle
Korie E. Handwerger1,2,*,
Zheng'an Wu1,*,
Christine Murphy1 and
Joseph G. Gall1,
1 Department of Embryology, Carnegie Institution of Washington, 115 West
University Parkway, Baltimore, MD 21210, USA
2 Department of Biology, Johns Hopkins University, 3400 North Charles Street,
Baltimore, MD 21218, USA
* These authors contributed equally to this study
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Fig. 1. Effect of heat shock on chromosome morphology. (A) Phasecontrast image of
one of the 18 bivalent chromosomes from an oocyte heat shocked for 4 hours at
31.5°C and allowed to recover for 3 hours at 18°C. The overall
contraction of the chromosome and loss of lateral loops is typical of
transcriptionally inactive chromosomes. (B) Immunofluorescent image of a
chromosome from an oocyte heat shocked for 4 hours at 31.5°C and allowed
to recover for 21 hours at 18°C. Pol II transcription has returned to
normal levels as shown by overall lengthening of the chromosome and
re-extension of the lateral loops. As in control preparations, chromosome
loops are specifically stained with mAb H14, which recognizes the largest
subunit of Pol II when the C-terminal domain is phosphorylated on serine
5.
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Fig. 2. A small fraction of the GV contents from a heat-shocked oocyte, spread on a
microscope slide and stained with mAb H1 against Xenopus coilin. (A)
Immunofluorescence to show the distribution of coilin. In this field there is
a single small CB (star) and eight mini-CBs. The whole preparation would
contain 50-100 large CBs, up to 8-10 µm diameter, and hundreds of
additional mini-CBs. (B) The same field by differential interference contrast
(DIC). The small CB (star) is associated with an unstained B-snurposome (b) of
similar size. The mini-CBs (arrowheads) occur free or attached to the surface
of B-snurposomes. Six extrachromosomal nucleoli (n) are also present in this
field.
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Fig. 3. Confocal images of mini-CBs (arrowheads) stained with four antibodies that
recognize typical CB components (first column) and counterstained with
 -coilin serum C236 to identify the mini-CBs (second column). The
overlay is shown in the third column and a DIC image is shown in the fourth
column. In each case, the mini-CB is attached to the surface of a B-snurposome
(b). (A) mAb H14, which recognizes the largest subunit of Pol II when the
C-terminal domain is phosphorylated on serine 5. Only the mini-CB is stained.
(B) mAb K121 against the trimethylguanosine cap found on several snRNAs stains
the mini-CB and two B-snurposomes. (C) mAb Y12 against the Sm epitope on
snRNPs stains the mini-CB and several B-snurposomes. (D) mAb No114 against
Xenopus Nopp140 stains the mini-CB and the nucleolus (n) but not the
B-snurposome.
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Fig. 4. A small fraction of the contents of a single GV from an oocyte that was
heat shocked at 31.5°C for 3 hours, injected with GFP-coilin transcripts
(into the cytoplasm) and allowed to recover overnight. (A) Immunofluorescence
after staining with an anti-GFP antibody to show the localization of
GFP-coilin in mini-CBs. The anti-GFP antibody enhanced the detectable but weak
signal from mini-CBs. (B) DIC image of the same field. Mini-CBs are indicated
by arrowheads. The larger round objects are B-snurposomes; a single nucleolus
(n) is also present.
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Fig. 5. Polyacrylamide gel electrophoresis of GV proteins from control oocytes
(-hs), oocytes heat shocked at 31.5°C for 4 hours (+hs) and oocytes
allowed to recover at 18°C for 12 hours after heat shock (hsR). All
oocytes were injected with [35S]-methionine to label newly
synthesized proteins. (A) Proteins stained with Coomassie Blue to show equal
loading of lanes and the absence of obvious changes in overall protein
composition. (B) Autoradiograph of the same samples showing marked reduction
in new protein synthesis after heat shock (lane 5) compared with control (lane
4), and renewal of protein synthesis during recovery from heat shock (lane 6).
Stars indicate regions of the gel where differences are detectable between the
control (lane 4) and heat shock recovery (lane 6). Molecular weight markers
are in kDa.
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Fig. 6. Western blot of three phosphorylated proteins from GVs of control oocytes
(-hs), oocytes heat shocked at 31.5°C for 4 hours (+hs) and oocytes
allowed to recover overnight at 18°C after heat shock (hsR). Immunostained
with mAb ARNA-3 against the largest subunit of RNA PolII, mAb No114 against
Xenopus Nopp140 and mAb H1 against Xenopus coilin. No
obvious differences in mobility were observed that might have indicated a
change in phosphorylation state.
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Fig. 7. Formation of mini-CBs in the absence of U7 snRNA. Confocal images of CBs
stained with mAb K 121 against the trimethylguanosine cap (first column) and
counterstained with -coilin serum C236 to identify the mini-CBs (second
column). The overlay is shown in the third column and a DIC image is shown in
the fourth column. (A,B) Mini-CB (arrowheads in A) and typical CB (cb in B)
from an oocyte that was heat shocked at 31.5°C for 4 hours and allowed to
recover overnight. The mini-CB and the typical CB stain strongly for both
trimethylguanosine (green) and coilin (red). The mini-CB is closely associated
with a B-snurposome (b). Nucleolus (n). (C) Mini-CB (arrowheads) and typical
CB (cb) from an oocyte that was heat shocked after injection of an
oligodeoxynucleotide against U7 snRNA. Elimination of U7 causes a marked
reduction in trimethylguanosine staining of CBs, because U7 is the major
capped snRNA in CBs. Mini-CBs are induced by heat shock in U7-depleted
oocytes; they too are deficient in trimethylguanosine staining (arrowheads in
C). Neither the morphology nor coilin staining of CBs is affected by U7
depletion.
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© The Company of Biologists Ltd 2002
