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FIG. 1. Nucleotide sequence of cloned histone HlA genomic DNA. Differences in the nucleotide sequence compared to the published sequence
(Perry et al., 1985) are underlined. Position 154-155, GC instead of CG; positions 333-338,464-466, 600- 647,793, insertions into the published
sequence: position 941. T instead of C.
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FIG. 2. Western blot analysis and quant ification of histone HlA
protein levels during early Xenop!t.' development. (A) Comigration of
HIA protein from XetW}'J'tu; gastrula embryos (XE) with HlA from
Xei'!Qpu.S liver (XL) and with in vitro synthesized HlA (i .v.). (B) Phos·
phor-Imager evaluation of a Western blot analysis of HlA in early
Xenrypu$ embryos. Total protein extract of three embryo equivalents
was loaded per lane, except for gastrula (0.5 embr-yo equivalents). Developmental
s tages according to Nicuwkoop and Faber {1967) arc indicated
in parentheses. /11 tritro synthesized (i.v.} HlA protein (110 pg)
served as the reference standard for quantificatiQn. (C) HlA accumulation
during gastrulation. Western blot as in B, except that detection
was by X ·ray film. Developmen tal stages are indicated with respect to
time after fertilization. The beginning of gastrolation was at 9 hr.
Three embryo equivalents were loaded for the 9·hr extract, one embryo
equivalent each for the other extracts.
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FIG. 3. Western blot analysis of Xenopus H IM protein. (A) HIM levels dur ing oogenesis and early development. Total protein extract of three
oocyte (egg or embryo) equivalents was l11aded per lane, except for s tage I -II oocytes (I-ll) and stage III- IV oocytes (III-IV) "'here the amount
of protein per lane was adjusted to match that of three s tage VI vocytes (VI). Oocyte stages are numbered according to Dumont (1972);
embryonic stages (arabic numerals) are according to Nicuwkoop and Faber (I967). VI mat, -in 11itmmatured oocytes; egg, unfertilized eggs; retic,
in ·vit-ro synthesized HIM protein. Note the HIM polymorphism in oocytes and embryos derived from different females indicated by lines
marked with 1, 2, and 3. (B) Subcellular distribution of HlM in full-grown oocytcs. Stage VI OOC)â¢tes were manually enucleated and protein
extracts prepared from the n\lclei {gv) and the enucleated oocytes (cyt). For comparison, extracts were prepared from unmanipulated oocytes
(oo) and from manually defolliculated oocytes (oo, def). Extract equivalents of three oocytes, and of 3 or 20 nuclei were analyzed, as indicated
he low each Ia ne.
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FlG. 4. In situ. immunofluorescence analysis of HIM and HlA on paraffin-embedded XenO'}»J..~ egg and embryo sections. Sections were
incubated with 1:400 diluted anti-HIM antiserum (A) or 1:100 diluted anti-HlA antiserum (B) and stained with an FfTC-conjugated secondary
antibody. Left panels show antibody staining; right panels show count~rstaining of the same sections with DAPI. Egg, metaphase plate of an
unfertilized egg; st..6.5, anaphase chromosomes of a stage 6.5 cleavage embryo; st.S, anaphase chromosomes (top) and interphase nucleus
(bottom) of a stage 8 blastula embryo; st.lO, segment from the marginal zone of a stage 10 early gastrula embryo. Sections were photographed at
a magnification of 630X (egg, st.6.5, s t.8) or 400X (st.lO).
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FIG. 5. Western blot analysis using anti-HlM antiserum of HlM-t!xpressing
cultured cells. -, untransfected cells; +, HiM-expressing
cells. Equal amounts of total protein were l<laded per lane. Molecular
weight markers are indicated in kDa.
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FIG. 6. In situ immunofluorescence analysis of HlM·expressing cells. B3.2 cells were transfectcd with the HIM expression plasmid pCMVB4.2,
fixed with methanol, and ~nin~nh~t.M with hnt.h OA PJ ~nd ant.i-Hl M ant.i~P.rum (1:400 rlilutP.d), follower! by Hl M dP.tection with an
FITC-coojugated secondary antibody. (a) Cell in interphase; (b) cell in mitosis; (c-e) different examples or HlM-positive cells exhibiting
aberrant chromatin condensation. Identical fields were photographed at a magnification of 630X to visualize either FITC (HlM) or DAPI
fluorescence.
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FIG. 7. Competition of Hl.M with Hl A. 83.2 cells were t ransfected with the HIM expression plasmid pCMV-84.2, fixed with paraformaldehyde,
and stained with DAPl and anti-HlA antiserum, 1:50 diluted, followed by detection with an FITC-conjugated secondary antibody. Cells
with aberrantly e.ondensed chromatin (H IM-positive cells) were identified by their DAPI fluorescence and photographed at a magnification of
400X both in the DAPI channel and in the FITC channel (Hl A). The coverslip was then removed and the cells stained for HlM with anti-HIM
antiser um, 1:200 diluted, followed by detection with an FITC-conjugatcd secondary antibody, and the same field was rephotographed (H1A +
HlM). Arrows in the HlA panels point to the HIM-expressing cells.
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FIG. 8. In situ immunofluorescence analysis of histone HlA-overexpressing cells. B3.2 cells were transfected with the expression plasmids
pCMV-HlA.7 (a, b, e) or pCMV-HlAA (c, d), fixed with paraformaldehyde, and incubated with DAPI and anti-HlA antiserum (1:200 diluted),
followed by detection with an FITC-conj ugated secondary antibody. Identical fields were photographed at a magnification of 630X to visuali~e
either FITC (HlA) or DAPI fluorescence. Photographic exposure was adjusted such that only transfected cells overexpressing histone HlA
were detectable in the FITC channel: staining intensities of HlA-overexpressing cells from different panels should not be directly compared
since different exposure times were used.
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