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Fig. 1. Fibrillarin mRNA and U3 snRNA accumulation during oogenesis. (A) Northern blot. Fib - Total RNA equivalent to one oocyte was separated by electrophoresis in a denaturing-formaldehyde 1 % agarose gel, electrotransferred to a nitrocellulose filter and hybridized under stringent conditions with a nick-translated Xenopus fibrillarin cDNA as a probe. The slight variations of electrophoretic mobility are due to the large amount of rRNA after stage II. U3 - Total RNA equivalent to five oocytes was separated by electrophoresis in a denaturing 7 M urea-5% polyacrylamide gel, electrotransferred on a nylon filter and hybridized under stringent conditions with a 3' labeled 22-mer synthetic oligonucleotide complementary to U3 snRNA (nt 101-122) U3 snRNA was identified using CHO 32P RNA as markers. The 'smile' of the gel is artefactual. (B) Quantitative data. Relative expression of fibrillarin mRNA, U3 snRNA, S8 rp-mRNA and rRNA during oogenesis. Upper panel: values, obtained by densitometric quantitation of the experiment shown in A and of others, are expressed relative to the highest point. Fibrillarin mRNA ( • •); U3 snRNA (O O) Lower panel already published curves for rRNA (solid line), r-protein S8 mRNA (long broken lines); nucleolin mRNA (short broken lines).
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Fig. 2. Expression of fibrillarin antigen during oogenesis Total protein extracts from ten defolliculated oocytes were prepared as descnbed in Materials and methods. Proteins corresponding to five oocytes were run in each lane of a 12 % SDS-PAGE. The gel was blotted onto nitrocellulose and probed with anti-fibrillarin serum. Detection of the complexes was achieved by a second antiserum coupled to alkaline phosphatase The insert shows a typical western blot, hp, hepatocyte nuclei; I to VI oocyte stages according to Dumont (1972). The graph shows quantitative data obtained by densitometnc scanning of the western blots of different experiments.
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Fig. 3. Nuclear localization of fibrillarin and U3 snRNA in Xenopus laevis oocyte (A) Indirect immunofluorescence microscopy on frozen sections of Xenopus ovary (a) Using the autoimmune antiserum against fibrillarin. Note the bright fluorescence of the amplified nucleoli as early as stage II (stages indicated in b). (b) Corresponding DAPI fluorescence. The nuclei of the follicule cells are stained (B) Western blot (Fib). Protein extracts were prepared from the different fractions as described in Materials and methods and analyzed by electrophoresis on a 12% SDS-PAGE. The gel was blotted onto nitrocellulose and probed with antiserum against fibrillarin. Detection of the complexes was achieved by 125I-proteinA. hp, Xenopus hepatocyte nuclei; T, total fraction of stage VI oocytes; C, cytoplasmic fraction; N, nuclearfraction (C) Northern blot (U3). RNA was prepared from the different fractions as described in Materials and methods and electrophoresed on a denaturing 7M urea - 5% acrylamide gel. After electrotransfer onto nylon filter, hybridization under stringent conditions was carried out with a 3' labeled synthetic oligonucleotide complementary to U3 snRNA (nt101-122). hp, Xenopus hepatocyte nuclei; T, total fraction of stage VI oocytes; C, cytoplasmic fraction; N, nuclear fraction
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Fig. 4. Fibrillarin mRNA and U3 snRNA accumulation during embryogenesis (A) Northern blot. Fib - Total RNA equivalent to one embryo was separated by electrophoresis on a denaturing-formaldehyde 1 % agarose gel,electrotransferred on a nitrocellulose filter and hybridized with a nick-translated Xenopus fibrillarin cDNA as a probe. U3- Total RNA equivalent to one or ten embryos was separated by electrophoresis on a denaturing 7 M urea-5 %polyacrylamide gel, electrotransferred to nylon filter and hybridized with a 3' labelled 22-mer synthetic oligonucleotide complementary to U3 snRNA (nt 101-122) hp, Xenopus hepatocyte nuclei, VI, stage VI oocytes; 0 to 40, stages of embryogenesis. (B) Quantitative data. Relative expression of fibrillarin mRNA, U3 snRNA, S8 rp-mRNA and rRNA during oogenesis. Upper panel: values, obtained by densitometric quantitation of the several experiments, are expressed relative to the highest point. Fibrillarin mRNA ( • • ); U3 snRNA (O O). Lower panel- already published curves for rRNA (solid line); r-protein S8 mRNA (long broken lines), nucleolin mRNA (short broken lines)
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Fig. 5. Localization of fibrillarin in Xenopus embryos. Indirect immunofluorescence staining, using antifibrillarin antibodies, of cryostat sections of embryos at different stages: control experiment, without the first antibody (A,B); blastula (C,D), late neurula; ectodermal cells (E,F); swimming tadpole (G,H). B,D,F,H, FITC images. A,C,E,G, corresponding DAPI images. Scale bars: in A,B,C,D,E,F=50um; G,H=100um The autofluorescence of yolk platelets is also observed in control (B).
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Fig. 6. Sedimentation of fibrillarin and U3 snRNA in glycerol gradients. Whole sonicates of stage 13 and stage 36 embryos were fractionated on 10-30 % glycerol gradients, and proteins and RNA extracted from gradient fractions as described in Materials and methods. FIB - western blot. Proteins were electrophoresed on a 12 % SDS-PAGE. The proteins were blotted onto nitrocellulose and probed with anti-fibrillarin serum. Detection of the complexes was achieved by 123I-protein A. Autoradiography was carried out for 20 h (stage 36) and for 72 h (stage 13). U3 - northern blot. RNA from different gradient fractions was electrophoresed on a denaturing 7 M urea - 5 % acrylamide gel, electrotransferred onto nylon filter (Hybond N) and hybridized with a 3' labelled synthetic oligonucleotide complementary to U3 snRNA (nt 101-122) Blots were exposed to X-ray films for 24 h (stage 36) and 36h (stage 13) hp, Xenopus hepatocyte nuclei; T, whole sonicate, 2 to 20, gradient fractions.
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