QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 January 2005
doi: 10.1242/jcs.01605

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Enukashvily, N. Articles by Podgornaya, O. Search for Related Content PubMed PubMed Citation Articles by Enukashvily, N. Articles by Podgornaya, O.

Satellite DNA binding and cellular localisation of RNA helicase P68

Natella Enukashvily^1,*, Rossen Donev^2,, Denise Sheer² and Olga Podgornaya¹

¹ Cell Cultures Department, Institute of Cytology, Tikhoretsky, 4, St Petersburg, 194064, Russia
² Human Cytogenetics Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (65K): [in a new window]   Fig. 1. Gel mobility shift assay (GMSA) of the nuclear and nuclear matrix extracts from mouse liver cells. Nuclear extract (NE, lanes 2-8) and nuclear matrix extract (NM, lanes 9-15) DEAE fractions (5 µg of total protein) were eluted from a DEAE-column by 0.2 M (lanes 2, 3, 9, 10), 0.3 M (lanes 4-6, 11-13) and 0.4 M (lanes 7, 8, 14, 15) NaCl. 1 ng of labelled MiSat fragment was added to the incubation mix. Each probe contained either 50-, 75- or 150-fold weight excess of E. coli DNA as indicated. The GMSA of the mock probe (without proteins added) is shown in lane 1. The fraction of the nuclear matrix eluted by 0.3 M NaCl was chosen for further work. The arrowheads point to the border between two sets of lanes (NE and NM).

View larger version (51K): [in a new window]   Fig. 2. GMSA of the 0.3 M NaCl mouse liver nuclear matrix fraction in the presence of different competitors. (A) E. coli DNA (50, 75, 150 weight excess, lanes 2, 3, 4, respectively), HS3 (lane 5), pUC19 (lane 6) and minor satellite (MiSat) (lane 7). HS3, pUC19 and MiSat were loaded in 50-fold weight excess. The mock probe GMSA is shown in lane 1. (B) The GMSA of the same fraction as in A in the presence of the competitors: dIdC, 100-fold weight excess to the labelled miSat probe (lane 2), pUC19 (lanes 3-5), MiSat (lanes 6-8), major satellite (MaSat) (lanes 9-11), telomere repeats (tel) (lanes 12-14), pUC 19 containing alphoid DNA monomer () (lanes 15-17), pUC19 with alphoid DNA 14-mer insertion (x14) (lanes 18, 19). All the competitors (except dIdC) were loaded in 50-(lanes 3, 6, 9, 12, 15, 18), 75-(lanes 4, 7, 10, 13, 16, 19) and 150-fold (lanes 5, 8, 11, 14, 17) weight excess. The mock probe GMSA is shown in lane 1.

View larger version (85K): [in a new window]   Fig. 3. Affinity purification of p68 on streptavidin-coated magnetic beads with biotinylated MiSat fragment. The fractions were subjected to 12% (A) or 10% (B) SDS-PAGE and stained with Coomassie Blue. (A) Lane 2, NM proteins; lane 3, NM proteins washed from the beads by 0.5 M NaCl in GMSA buffer; lane 4, proteins washed from the beads by boiling in SDS-PAGE sample buffer. (B) Influence of competitor DNA (MiSat, lane 3; pUC19, lane 4) on the eluate composition. The probes in lane 4 contained 50-fold excess of pUC19, in lane 3, 10-fold weight excess of MiSat containing p238 as competitor DNA when loaded into affinity column. The nuclear matrix extract loaded is shown in lane 2. P68 and lamin B (L) bands are indicated. The binding activity of fractions in GMSA is indicated at the top. Size markers are shown in lane 1 of panels A and B.

View larger version (42K): [in a new window]   Fig. 4. Western blotting of the GMSA nuclear matrix extract and DEAE-purified fractions. Coomassie Blue staining (1-3) and immunoblot (4-9) of the nuclear matrix extract (lanes 2, 4, 6, 8) and 0.3 M NaCl DEAE fractions (lanes 3, 5, 7, 9) detected with antibodies against P68, lamins A/C or lamin B as indicated.

View larger version (23K): [in a new window]   Fig. 5. Computer analysis of the amino acid sequence of P68. (A) Results of potential coiled-coil domain search using COIL software. x-axis shows the amino acid residues starting from N-terminus, y-axis, the coiled-coil formation probability, P. (B) Computer alignment of human/mouse P68 (h/m P68) with mouse (mLB) and human (hLB) lamins B2 rod domain fragments. Potential coiled coils shown in A are boxed in yellow (P=0.62) and in orange rectangles (P>0.95). The green rectangle is coil 1A, the blue rectangle is coil 1B. The line below the alignment shows the similarity score: *, amino acids in that column are identical;:, conserved substitutions;., semi-conserved substitutions. The lines labelled p68 PrSS and LB PrSS represent prediction of the secondary structure of P68 and lamin B2, respectively, from its primary sequence. H, a region of -helical structure; E, extended sheet; _ flexible coil structure.

View larger version (67K): [in a new window]   Fig. 6. ImmunoFISH of HeLa cells fixed with 2% PFA for 30 minutes. Antibodies against P68 (green) and alphoid DNA (A) and HS3 (B) probes (in red) were used to probe HeLa cells. (C) Double immunofluorescence staining of HeLa cells with SC35 antibody (red) and the antibody against P68 (green). Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 7. Immunofluorescent staining of synchronised mouse L929 cells with antibody against P68. Cells were stained with anti-P68 (green) and counterstained with DAPI (red pseudocolour) to reveal the location of p68 with regard to chromocentres, and were captured in G1 (A), early S (B), late S (C), G2 (D). The first column shows merged images. Bar, 10 µm.

View larger version (74K): [in a new window]   Fig. 8. Double immunofluorescence staining of mouse L929 cells with antibodies against P68 and SC35. P68 (green) and SC35 (red) staining of cell nuclei at G1 (A) and G2 (B) with merged images in the first column. Bar, 10 µm.

View larger version (54K): [in a new window]   Fig. 9. Double immunofluorescence of SC35 and centromeric protein in L929 cells. Cells in G1 show the spatial proximity of some centromeres stained with antibody against CENP-B (III, red) to SC35 domains (II, green). Cells were counterstained with DAPI (IV, blue). Merged image (I) Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 10. ImmunoFISH of L929 mouse cells at various cell stages. Cells at G1 (A), early S (B), late S (C), late G2-early prophase (D) were used for colocalisation studies with P68 and FISH with a MiSat probe. Column I, merged image (P68 in green, centromeric proteins in red); II, staining with P68 antibody; III, FISH with MiSat probe; IV, DAPI staining (light blue). Bar, 10 µm.

View larger version (108K): [in a new window]   Fig. 11. Immunofluorescence of L929 mitotic chromosome spreads. Cells spreads were fixed with methanol:acetic acid (A), with paraformaldehyde (B) and prematurely condensed G2 chromosome preparation (C). P68 is shown in green. Chromosomes were counterstained with DAPI and pseudocoloured in red. Centromeres stained with an antibody against P68 are indicated with white arrowheads; the interchromosome connection is indicated with black arrowheads. Bar, 20 µm.