XB-ART-28194J Cell Biol 1987 Apr 01;1044:841-7.
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Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis.
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 tadpole parvalbumin 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 tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.
PubMed ID: 3558484
PMC ID: PMC2114428
Species referenced: Xenopus laevis
Genes referenced: mindy3 ocm3 ocm4.2 ocm4.5
Antibodies: Ocm Ab1
Article Images: [+] show captions
|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 tadpole tail muscle cells. Strips of tail muscle 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 tail muscle 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.|
References [+] :
Berchtold, Primary structure of parvalbumin from rat skeletal muscle. 1983, Pubmed