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Fig. 1. Immunofluorescence microscopy showing reactions of monoclonal antibody VIM-3B4 to vimentin on cultured kidney
epithelial (XLKE) cells of line A6 (A-C) and a frozen section of ovary tissue of Xenopus laevis (D,E). (A) Typical fibrillar
networks of vimentin IFs are seen in XLKE-A6 cells. (B,C) Epifluorescence (B) and phase-contrast (C) pictures of the same
field of an XLKE-A6 cell monolayer after treatment with 10 M-colcemid for 4h, showing aggregates of collapsed IF in the
perinuclear cytoplasm, as is typical for vimentin IF. (D,E) Epifluorescence (D) and phase contrast (E) pictures of a cryostat
section of ovarian tissue, showing bright staining of interstitial cells as well as endothelial cells and erythrocytes of a blood
vessel. Bars, 50,um.
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Fig. 3. Identification of the polypeptides encoded by the
cDNA clones pXenViml and pXenVim4. Coelectrophoresis
of the high-salt-buffer-resistant cytoskeletal proteins of
XLKE-A6 cells (same system and symbols as in Fig. 2C,D)
with products of in vitro translation from hybrid-selected
embryonal stage-18 mRNA (A,B) and from mRNA
obtained by in vitro transcription/translation of pXenViml
(C,D). Coelectrophoresis of the mixed products obtained
from in vitro transcription/translation of pXenViml and
pXenVim4, respectively (E,F). (A,B) Coomassie-brilliantblue-
stained gel (A) and corresponding autoradiograph (B),
showing that the [ SJmethionine-labelled in vitro
translation products from hybrid-selected mRNA comigrate
with unlabelled vimentin (bracket) of XLKE-A6 cells.
(C,D) Coomassie-brilliant-blue-stained gel (C) and
corresponding autoradiograph (D) of the product obtained
by in vitro transcription and translation, showing that the
[ SJmethionine-labelled translation product comigrates with
unlabelled vimentin (bracket) of XLKE-A6 cells. Note that
some modification, probably phosphorylation, also takes
place in the rabbit reticulocyte assay used for in vitro
translation, resulting in the appearance of a minor, more
acidic variant. (E,F) Coomassie-brilliant-blue-stained gel
(E) and corresponding autoradiograph (F) of mixed in vitro
transcription/translation products of Xenopus vimentin
clones pXenViml and pXenVim4. As revealed from
parallel gels of individual [35S]methionine-labelled
translation products, pXenVim4 is translated into a
polypeptide slightly more acidic and less mobile (triangle)
than pXenViml (bracket). Fluorography was for 4h. After
prolonged exposure acidic variants were also visible.
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Fig. 4. Nucleotide sequence and deduced amino acid sequence of cloned Xenopus laevis vimentins. (A) Combined sequence
of clones pXenViml, containing nucleotides 1-1688 (indicated by an arrow), and pXenVim3, containing nucleotide 1367
(indicated by an arrowhead) to the 3'-end of the clone. The clones are identical in the overlapping region. Differences found
in the pXenVim2 sequence are indicated by an upward triangle (insertion of an AGC triplet coding for serine) and a
downward triangle (substitution of T for C without change of the coded amino acid). (B) Sequence of clone pXenVim4.
Major differences of the nucleotide sequence to pXenViml are overlined and the amino acid changes are encircled.
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Fig. 5. Amino acid sequence comparison of Xenopus vimentin Vim 1 (Xen), chicken vimentin (Chk; taken from Zehner et
al. 1987) and hamster vimentin (Ham; taken from Quax et al. 1983). Bold-faced letters denote amino acids identical in
Xenopus and at least one of the other two species. Amino acid sequences have been aligned for maximal homology,
insertions introduced for this purpose are denoted by horizontal bars. The downward arrow demarcates the start and the
upward arrow the end of the a'-helical rod domain. The dots represent positions a and d of the heptade convention to
maximize coiled-coil configuration. The rod domain contains two non-o--helical interruptions of 11 and 43 amino acids,
respectively, giving rise to coiled-coil subdomains 1A (CIA), IB (C1B) and 2 (C2). The arrowhead indicates an alteration in
the heptade pattern that probably results in a 'stutter' in coil 2.
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Fig. 6. Comparison of the 3'-untranslated sequences of vimentin mRNAs of Xenopus (Xen), chicken (Chk; from Zehner et
al. 1987), hamster (Ham; from Quax et al. 1983) and human (Hum; this study) as determined from cDNAs and genes,
respectively. Sequences have been aligned for maximal homology and insertions introduced for this purpose are denoted by
horizontal bars. The three stars mark the ends of the coding region. Regions of relatively high homology are boxed and
numbered by roman numerals. Bold-faced letters denote nucleotides present in all four sequences. The apparent consensus
sequences are indicated underneath each box; Lower case letters indicate the presence of at least three identical nucleotides.
P, pyrimidine nucleotide; R, purine nucleotide. The arrowheads indicate the probable polyadenylation sites in the genomic
sequences for chicken (Zehner & Paterson, 1983) and hamster vimentin (Quax et al. 1983). Note that the presumptive
polyadenylation signal (overlined) in box VII is flanked by a highly conserved sequence.
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Fig. 7. Detection of vimentin-specific mRNA in various
tissues and cells of adult animals and in embryonic stages of
Xenopus laevis by RNA blot analysis (A-D) and RNase
protection assays (E-H). (A) RNA was prepared from
various tissues (Davis et al. 1986), and 20 //g total RNA
(lanes 1, 4 and 5) or 1 fig poly(A)+RNA (lanes 2, 3, 6 and
7) were applied for formaldehyde/agarose gel
electrophoresis. Hybridization was carried out with a
random-primed, 32P-labelled (O-SxlO^tsmin"1 ml"1), 5'-
end-specific probe of pXenViml. Lane 1, oesophagus; lane
2, XLKE-A6 cells; lane 3, skin; lane 4, skeletal muscle;
lane 5, cardiac muscle; lane 6, liver; lane 7, ovary.
Horizontal bars indicate the positions of Xenopus 28 S and
18 S rRNA. Exposure: 24 h. (B) RNA was prepared from
ovary of adult frogs and embryos of the indicated
developmental stages, 20 ^g total glyoxylated RNA were
loaded per lane. RNA was blotted to Genescreen plus and
hybridized with a 32P-labelled (3xl06ctsmin~1 ml"1) antisense
RNA probe from pXenViml. The final washes were
with 0-lxSSC, 0-5% SDS at 72°C. Lane 0, ovary; lane 1,
unfertilized eggs; lane 2, stage 6-5 (morula); lane 3, stage 9
(fine cell blastula); lane 4, stage 11 (gastrula); lane 5, stage
14 (neural plate stage); lane 6, stage 18 (neural groove
stage); lane 7, stage 28; lane 8, stage 39; lane 9, stage 42
(swimming tadpole). Horizontal bars indicate the positions
of bovine 28 S, E. coli 23 S and bovine 18 S rRNAs.
Exposure: 4h. Because of the higher activity of the
riboprobe, the signal obtained for ovarian tissue appears
enhanced, compared to that shown in A, lane 7). (C) RNA
as loaded in B was run on a formaldehyde/agarose gel,
blotted to nitrocellulose and hybridized with P-labelled
probe as in B. The final washes were with 0-lxSSC, 0-1 %
SDS at 65°C. Lane 1, unfertilized eggs; lane 2, stage 6-5;
lane 3, stage 9; lane 4, stage 11; lane 5, stage 14; lane 6,
stage 18; lane 7, stage 28; lane 8, stage 39; lane 9, stage 42.
Horizontal bars indicate the positions of Xenopus 28S and
18S rRNA. Exposure: 5h. After 64 h exposure, the signals
were considerably enhanced as shown in lanes 1' and 2'
which correspond to lanes 1 and 2. Note the reaction with
18S and 28S rRNAs. (D) Blot shown in C after
ribonuclease A treatment (20/igmn1 for 20min at 25°C
followed by 65 °C washes with OlxSSC, 01 % SDS).
Exposure: 10 days. Note that the signal at the position of
the rRNA disappeared whereas that at the position of
vimentin mRNA persisted (lanes 2-9). (E) Autoradiogram
of a ribonuclease protection assay with 1 /ig sense RNA
generated by in vitro transcription from pXenViml (lanes
10 and 12) and from the cytokeratin clone pXenCK 1/8
(lanes 9 and 11), showing that the sense RNA of
pXenViml, but not that of pXenCKl/8, specifically
protects the 32P-labelled anti-sense vimentin probes (arrows
in lanes 10 and 12). Hybridizations shown here were carried
out at 45°C (lanes 1, 2, 7 and 8) or 60°C (all other lanes)
for 15h with lxlO5 ctsmin"1 (~lfmole) of the uniformly
labelled 3'-specific probe (lanes 2, 4, 6, 8, 11 and 12) or the
'TYRKLEGE-probe' (see Materials and methods; lanes 1,
3, 5, 7, 9 and 10). To control for stringency, the probes
were hybridized with 10 fig tRNA (lanes 1-8) and either
subjected to RNAse treatment (lanes 5-8) or processed
further without RNAse treatment (lanes 1-4). Lane M
shows f/pall-restricted pBR 322 size markers (from top to
bottom: 622, 527, 403, 309, 242, 238, 217, 201, 190, 180,
160, 147, 123, 110, 90 nucleotides). Exposure: lh.
(F) Autoradiogram of a ribonuclease protection assay with
5 /Ig total Xenopus RNA from unfertilized eggs (odd
numbers) and stage 18 (even numbers) using the 3'-specific
probe (lanes 1-4) or the 'TYRKLEGE probe' (lanes 5-8).
Hybridization was at 45°C (lanes 3, 4, 7 and 8) or 60°C
(lanes 1, 2, 5 and 6). Arrows mark the fully protected
probes without polylinker sequences. The arrowhead in
lane 4 marks a prominent protected fragment of 160
nucleotides. Exposure: 3 days. (G) Autoradiogram of a
ribonuclease protection assay with 5 /tg total RNA from
oocytes (lane 1), unfertilized eggs (lane 2), stage-6-5 (lane
3), stage-18 (lane 4) and stage-28 embryos (lane 5)
hybridized to the 3'-specific probe at 65°C. Exposure: 3
days. (H) Autoradiogram of a ribonuclease protection assay
with 5/<g total RNA from oocytes (lane 1), unfertilized eggs
(lane 2), stage-6-5 (lane 3), stage-9 (lane 4) and stage-28
embryos (lane 5) hybridized to the 3'-specific probe at 65°C
in buffer containing only 50 % formamide as compared to
80% as used in panels E-G. Exposure: 3 days.
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Fig. 8. Immunofiuorescence microscopy performed on cryostat sections of frozen ovarian tissue from Xenopus laevis using
monoclonal antibody VIM-3B4. (A,B) The same field is shown in epifluorescence (A) and phase-contrast (B) optics. Note
that only the interstitial cells show a bright fluorescence, whereas the oocytes are negative. (C,D) In a region with
previtellogenic oocytes (C, epifluorescence; D, phase contrast) the oocytes are negative whereas the interstitial cells, the
endothelial cells and the erythrocytes of the blood vessel show intense fluorescence, v, blood vessel. Bars. 50um.
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Fig. 9. Survey picture montage of
immunofluorescence micrographs of
sections through snap-frozen stage-
14 embryos (neural plate stage) of
Xenopus laevis (whole embryos
were sectioned and documented in
overlapping micrographs).
(A) Reaction of monoclonal
antibody VIM-3B4, showing
positive reaction only in a distinct
and thin mesenchymal cell layer
(demarcated by brackets) whereas
the neural plate (np), notochord
(no) and somites (s) are negative.
ec, ectoderm; en, endoderm.
(B) Reaction of monoclonal
cytokeratin antibody lu-5, showing
an area corresponding to that
shown in A in a step section. Note
that all cell layers are positive for
cytokeratins. In particular, the
notochord (no) and the ectoderm
(ec) are intensely stained. The
mesenchymal cell layer (brackets)
shown to be positive for vimentin in
A, is also positive for cytokeratin.
Symbols are as in A. Bars, 50,um.
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Fig. 10. Immunofluorescence microscopy performed on cryostat sections of snap-frozen stage-18 (neural groove stage)
embryos of Xenopus laevis using monoclonal antibodies to vimentin (A) and cytokeratin (B). (A) Immunofluorescence
micrograph of the neural groove region of a stage-18 embryo, showing the reaction of monoclonal antibody VIM-3B4. The
reaction is restricted to a single cell layer (brackets) beneath the neural groove (n). All other cell layers are negative.
ec, ectoderm; s, somite; n, neural groove; no, notochord. (B) Reaction of monoclonal antibody lu-5, on a step section of the
same region shown in A. Note that all cell layers show a positive reaction, the notochord (no) and the ectoderm (ec) being
brightly stained and that the thin layer of cells shown to be positive for vimentin in A is also positive for cytokeratin
(brackets). Symbols are as in A. Bars, 50 um.
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Fig. H- Analysis of various fractions from oocytes and unfertilized eggs of Xenopus laevis for the presence of vimentin by SDS-PAGE and immunoblotting with different
antibodies. (A) Proteins transferred to nitrocellulose were
stained by Ponceau S after the alkaline phosphatase
immunostaining reaction shown in B. Lane 1, proteins recovered in the 100 000 g supernatant from total egg
extracts (HSS proteins), concentrated by precipitation with
acetone, 30-egg equivalents; lane 2, HSS proteins directly loaded, 12-egg equivalents; lane 3, high-salt-buffer-resistant
« a fraction of the low-speed pellet from egg homogenates; lane
" 4, insoluble fraction from high-salt-buffer-extracted material
1 2 3 4 5 6 7 1 2 3 4 5 6 7 pelleted at lOOOOg after an initial 800g centrifugation; lane
5, high-salt-buffer-resistant material pelleted at 800g; lane
6, high-salt-buffer-resistant material pelleted at lOOOOg.
Lanes 3-6, 20-egg equivalents. The prominent high
molecular weight band in lanes 3-6 contains the abundant
major yolk protein. Lane 7, total cytoskeletal proteins of
XLKE-A6 cells, 2 fig, representing approximately 0-2 ng
vimentin (see Fig. 2A). (B) Immunoreaction corresponding
to A, developed until background staining appeared, as
shown by the unspecific reaction of the Mr —100000 yolk
protein band (lanes 3-6) which was also seen when blots
were only incubated with second antibody. Specific
vimentin reaction is only seen in XLKE-A6 cells (lane 7).
(C,D) Calibration of the sensitivity of antibody VIM-3B4
for the detection of Xenopus vimentin. (C) For
quantification, Xenopus vimentin synthesized in E. coli
transfected with clone pXenViml (cf. Magin et al. 1987)
was dissolved in electrophoresis sample buffer, and a 10 uL
sample was applied to the gel (lane 2). For comparison,
bovine serum albumin was loaded as follows: lane 1, 1-4 fig;
lane 3, l-2^g; lane 4, l^g; lane 5, 0-8jUg; lane 6, 0-6f«g;
lane 7, 0-4//g. As estimated after staining with Coomassie
brilliant blue, the applied amount of vimentin corresponded
to approximately l^g of bovine serum albumin.
(D) Immunoblot analysis of recombinant Xenopus vimentin
diluted to various degrees: lane 1, l^g; lane 2, 500 ng; lane
3, 100 ng; lane 4, 50 ng; lane 5, 10 ng; lane 6, 5ng; lane 7,
1 ng. Note faint reaction in lane 6, showing that 5 ng are
detectable. The band of somewhat higher SDS-PAGE
mobility is proteolytically trimmed vimentin of Mr ~38000.
Blots shown in B and D have been processed in parallel.
(E,F) Examination of cytoskeletal fraction from unfertilized
eggs with guinea pig antibodies to vimentin (E), in
comparison with reference vimentin (F).
(E) Autoradiogram of an immunoblot analysis of proteins
from 15 unfertilized eggs separated by two-dimensional gel
j - a l a electrophoresis, followed by incubation of nitrocellulose
blots with guinea pig antibodies to vimentin and 125Ilabelled
protein A. (F) Parallel experiment to that shown in
E with cytoskeletal proteins from Chinese hamster ovary fibroblasts containing approximately l,ug vimentin. Both blots were
exposed for 3 days at 70°C using intensifying screens. (G-L) Examination of the presence of vimentin in oocytes of Xenopus
by two-dimensional gel electrophoresis, followed by immunoblotting. (G) Cytoskeletal proteins from 150 mg oocytes were
separated by two-dimensional gel electrophoresis, transferred to nitrocellulose and stained with Ponceau S. The bracket
denotes the major oocytes type II cytokeratin l/8, the three arrowheads denote a triplet of unidentified cytoskeletal proteins
of Mr 56000-60000. Positions of cytokeratin 2 and 3, corresponding to human cytokeratin 18 and 19 and a group of basic
cytokeratins of Mr —56000 are also indicated. (H) The same amount of protein as shown in G was mixed with cytoskeletal
proteins from XLKE-A6 cells for the identification of the position of Xenopus vimentin (arrow) relative to the oocyte
proteins. Symbols as in G. (I) Immunoblot analysis of cytoskeletal oocyte proteins with monoclonal antibody PK VI. Note
absence of staining at the position of vimentin (arrows in H and J). Weak staining of five polypeptides of Mr 56000-60000 is
indicated by arrowheads. (J) Immunoblot analysis of a mixture of cytoskeletal proteins from oocytes and XLKE-A6 cells
with monoclonal antibody PK VI. Note staining of cytokeratin 1/8 and vimentin but not component X. (K) Immunoblot
analysis of cytoskeletal oocyte proteins with antibody anti-IFA. Note staining of cytokeratin 1/8 but absence of staining at
the position of vimentin. Note weak staining of triplet polypeptides of Mr 56000-60000. (L) Immunoblot analysis of a
mixture of cytoskeletal proteins from oocytes and XLKE-A6 cells with anti-IFA. Vimentin as well as several cytokeratins are
heavily stained, thus demonstrating that anti-IFA reacts with Xenopus vimentin. Comparison of L and K shows absence of
detectable vimentin in K.
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