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A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpoleparvalbumin is highly similar to previously characterized beta-parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpoletail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.
Figure 1. Gel electrophoretic analysis of proteins encoded by
recombinant ~,PVI. A displays the Coomassie Blue-staining pattern
oft 10% polyacrylamide-SDS gel, B represents a Western blot with
preimmune serum, C shows a similar blot with immune serum, and
D exhibits an autoradiogram of 45Ca2+ binding to filter-bound proteins.
In the four panels, the proteins synthesized by bacteria infected
by the vector kgtll are shown in lanes marked g, whereas
proteins produced by bacteria infected by ~,PV1 are shown in lanes
marked PV. The immune complexes on the filters were detected
with secondary goat anti-rabbit IgG antibody coupled to horseradish
peroxidase and chloronapthol, whereas the 4~Ca2+ binding was
detected by film autoradiography. Lane M contains molecular
weight standards with the molecular weights of the individual species
listed in kilodaltons on the left.
Figure 2. Nucleotide sequence of the LPV1 insert, a portion of pPV2 recombinant cDNA plasmid, and the predicted parvalbumin protein.
The insert of LPV1 was sequenced by the dideoxy method in both directions; the sequence is delineated with x's. The complete protein
sequence was deduced by sequencing rightward from the Pvu II site of the recombinant pPV2. A protein encoded by the longest open
reading frame in this sequence is shown, with a putative translation start site (Met) and the high affinity Ca2÷-binding loop domains (24)
of parvalbumin underlined, and the termination codon marked with asterisks. In this same interval, there are 6 and 11 translation stop
codons in each of the other two forward reading frames. The regions of homology between carp beta-parvalbumin (10) and the Xenopus
putative parvalbumin are shown below; identities and semiconservative changes between the two sequences (27) are noted by two and one
dots, respectively.
Figure 3. Western blot with rabbit anti-parvalbumin antibodies to
lysates from oocytes, eggs, and various Xenopus staged embryos.
The lanes (from left to right) contain extracts from 1/4 equivalents
of oocyte (stage VI), egg, stage 10 (gastrula), stage 16 (neurula),
stage 22, stage 24 (tailbud), stage 31, stage 41 (tadpole) embryos,
and dissected stage 41 head and tail segments. Proteins were
resolved by electrophoresis in a 20% polyacrylamide-SDS gel and
later transferred electrophoretically to nitrocellulose. Immune
complexes were detected with 125I-labeled goat anti-rabbit IgG antibodies
and film autoradiography.
Figure 4. Northern blot analysis
of parvalbumin homologous
RNAs present in oocytes,
eggs, and various staged embryos.
The blot was hybridized
with 32P-labeled parvalbumin
cDNA. The nucleotide
(nO lengths of some of the
pBR322 DNA Taq I restriction
fragments (lane M) are
shown on the right of the autoradiogram.
The lanes (from
left to right) contain extracts
from two oocytes (stage VI),
eggs, stage 10 (gastrulae), stage
16 (neurulae), stage 22, stage
37, and stage 41 embryos.
Figure 5. Immunological detection ofparvalbumin
in tadpoletailmuscle cells. Strips of tailmuscle from stage 41 tadpoles were permeabilized
with Triton X-100 and incubated sequentially
with anti-parvalbumin antibodies and
goat anti-rabbit IgG antibodies coupled to fluorescein.
A shows a phase contrast image of the
tailmuscle strip, whereas B shows the fluorescent
image. The black spots in A are epidermal
melanocytes. Bar, 30 um.
Figure 6. Immunofluorescent detection of parvalbumin in stage 28 developing muscle cells. A shows a stage 28 embryo, B displays an
isolated NNS complex from a stage 28 embryo, C shows an azure blue-stained squash of the NNS complex, D shows a phase contrast
image of the differentiating muscle cells, and E shows the fluorescence of these cells reacted with anti-parvalbumin antibodies. Bars: (A)
400 um; (C) 85 um; (D) 15 um.
Figure 7. Western blot detection
of parvalbumin in developing somites
of Xenopus embryos. Protein
lysates were prepared from
adult leg muscle, the NNS complex
of two stage 28 embryos,
the remainder of two stage 28
embryos minus their NNS complexes,
and one complete stage 28
embryo. The proteins were resolved
on a 18 % polyacrylamide-
SDS gel by gel electrophoresis
and blotted to nitrocellulose. The
antigen complexes formed with
rabbit anti-parvalbumin antibodies
were detected with 125I-protein
A and X-ray film autoradiography.
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