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First published online 25 February 2003
doi: 10.1242/jcs.00353

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A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription

¹ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, SE-17177 Stockholm, Sweden
² Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia

View larger version (47K): [in a new window]   Fig. 1. Predicted amino acid sequences of the two isoforms of the C. tentans p40/50 protein. (A) The upper sequence corresponds to the p40 isoform and the lower sequence corresponds to the p50 isoform. The sequences are identical for the first 258 amino acids. The predicted cold-shock domain is boxed. (B) Schematic presentation of the domain organisation of the two isoforms of the p40/50 protein.

View larger version (26K): [in a new window]   Fig. 2. Western blot analysis of the p40/50 protein. (A) Comparison of p40/50 in C. tentans salivary glands and tissue culture cells (total extracts). Recombinant p40 served as control. An anti-p40/50 serum was used as primary antibody and an alkaline phosphatase-conjugated antibody as secondary antibody. (B) Demonstration of p40/50 in nuclear and cytoplasmic extracts from C. tentans tissue culture cells. The whole nuclear extract and 1/10 of the cytoplasmic extract were loaded onto SDS-polyacrylamide gels and blotted. The blots were either immunostained using an affinity-purified p40/50 antibody followed by an alkaline phosphatase-conjugated antibody (a), or stained with Coomassie blue (b). The positions of molecular mass standards are shown to the left in kilodaltons.

View larger version (167K): [in a new window]   Fig. 3. Immunocytological localisation of p40/50 in C. tentans salivary gland cells. Semi-thin cryosections of salivary glands were prepared and immunostained with an affinity-purified antibody to p40/50 (A,A') or a pre-immune serum (B,B') as primary antibody and a gold-conjugated antibody as secondary antibody. The immunogold labelling was silver-enhanced and photographed both in bright-field (A,B) and phase-contrast (A',B') microscopy. n, nucleus; c, cytoplasm; nu, nucleolus. Bar, 20 µm.

View larger version (95K): [in a new window]   Fig. 4. Immunolocalisation of the p40/p50 protein on isolated chromosomes from C. tentans salivary gland cells. Polytene chromosomes were isolated and incubated with an affinity-purified p40/p50 antibody (A,B) or pre-immune serum (C,C' and D,D'), and subsequently incubated with a gold-conjugated secondary antibody. The immunogold labelling was silver-enhanced, and the specimens were photographed both in bright-field (A-F) and phase-contrast (C'-F') microscopy. Some chromosomes were RNase A treated before immunolabelling (E,E' and F,F'). A,A',C,C',E,E', chromosome IV; B,B',D,D'F,F', chromosome I. The three Balbiani rings (BR1-3) have been indicated in (A). Bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 5. Immunoelectron microscopic localisation of p40/50 in Balbiani rings (A), nucleoplasm (B) and cytoplasm (C) in salivary gland cells. Ultrathin cryosections of salivary glands were incubated with a p40/50 antibody, a gold-conjugated secondary antibody was added to visualise the labelled sites, and the sections were finally embedded in polyvinyl alcohol. Small arrows indicate the position of gold particles; the barred arrows denote specifically labelled BR particles. A schematic drawing outlining the assembly and transport of BR particles is shown below the electron micrographs. Large arrows relate the electron micrographs to the corresponding stage during mRNA biogenesis. Bar, 100 nm.

View larger version (94K): [in a new window]   Fig. 6. Immunoelectron microscopic localisation of p40/50 along a transcriptionally active BR gene. Polytene chromosomes were isolated, incubated with a p40/50 antibody and detected with a secondary antibody conjugated to 12 nm colloidal gold markers. (A) Section through a Balbiani ring showing several segments of active genes. Examples of proximal (p), middle (m) and distal (d) segments have been indicated. Arrows mark the position of gold particles. (B) Schematic drawing of an active BR gene. Growing particles in the promoter-proximal (p), middle (m) and promoter-distal (d) portions of the gene are shown, as well as the chromatin axis. (C-G) Immunolabelled proximal (C), middle (D,E) and distal (F,G). segments of BR genes. Schematic drawings are shown below the electron micrographs, with examples of labelled nascent RNPs (dark) and unlabelled RNPs (outlined). The chromatin axis is indicated by a short dashed line. Bars, 100 nm.

View larger version (36K): [in a new window]   Fig. 7. UV-crosslinking of p40/50 to nuclear and cytoplasmic poly(A) RNA in vivo. Nuclear (N) and cytoplasmic (C) extracts were prepared from both UV-irradiated (+UV) and control (–UV) tissue culture cells. The crosslinked proteins were collected with oligo(dT) chromatography, fractionated by SDS-PAGE and analysed by western blotting. Antibodies against p40/50 and hrp36 (positive control) were used in the western blot.