-
4
-
-
0025114466
-
-
K. Gottesdiener, J. Garcia-Anoveros, M. G. Lee, L. H. T. van der Ploeg, Mol. Cell. Biol. 10, 6079 (1990).
-
(1990)
Mol. Cell. Biol.
, vol.10
, pp. 6079
-
-
Gottesdiener, K.1
Garcia-Anoveros, J.2
Lee, M.G.3
Van Der Ploeg, L.H.T.4
-
5
-
-
0021215533
-
-
L. H. T. van der Ploeg, D. C. Schwartz, C. R. Cantor, P. Borst, Cell 37, 77 (1984).
-
(1984)
Cell
, vol.37
, pp. 77
-
-
Van Der Ploeg, L.H.T.1
Schwartz, D.C.2
Cantor, C.R.3
Borst, P.4
-
7
-
-
0025862186
-
-
M. Weiden, Y. N. Osheim, A. L. Beyer, L. H. T. van der Ploeg, Mol. Cell. Biol. 11, 3823 (1991).
-
(1991)
Mol. Cell. Biol.
, vol.11
, pp. 3823
-
-
Weiden, M.1
Osheim, Y.N.2
Beyer, A.L.3
Van Der Ploeg, L.H.T.4
-
9
-
-
0021767869
-
-
L. H. T. van der Ploeg, A. W. C. A. Cornelissen, J. D. Barry, P. Borst, EMBO J. 3, 3109 (1984).
-
(1984)
EMBO J.
, vol.3
, pp. 3109
-
-
Van Der Ploeg, L.H.T.1
Cornelissen, A.W.C.A.2
Barry, J.D.3
Borst, P.4
-
12
-
-
0025313211
-
-
H. M. M. Chung, C. Shea, S. Fields, R. N. Taub, L. H. T. van der Ploeg, EMBO J. 9, 2611 (1990).
-
(1990)
EMBO J.
, vol.9
, pp. 2611
-
-
Chung, H.M.M.1
Shea, C.2
Fields, S.3
Taub, R.N.4
Van Der Ploeg, L.H.T.5
-
15
-
-
0021971721
-
-
C. R. Birkett, K. E. Foster, L. Johnson, K. Gull, FEBS Lett. 187, 211 (1985).
-
(1985)
FEBS Lett.
, vol.187
, pp. 211
-
-
Birkett, C.R.1
Foster, K.E.2
Johnson, L.3
Gull, K.4
-
16
-
-
1842375356
-
-
note
-
For combined immunofluorescence and fluorescence in situ hybridization, procyclic trypanosomes (strain 427) were washed once in phosphate-buffered saline (PBS), transferred to slides coated with aminopropyltriethoxysilane (Sigma), and fixed with 4% formaldehyde-5% acetic acid in PBS for 15 min at 20°C. After washing twice with PBS, cells were permeabilized with 0.1% NP-40 in PBS for 5 min, washed once in PBS, and incubated with the first antibody in PBS for 1 hour, then washed three times for 5 min each in PBS and incubated with a secondary, fluorescein isothiocyanate (FITC)-conjugated antibody (Dako) for 45 min. After washing, cells were postfixed in 3% formaldehyde for 20 min. Cells were then prehybridized for 60 min in 50% formamide, 2× saline sodium phosphate-EDTA (SSPE), 10% dextran sulfate [hybridization buffer (HB)] at 37°C, and subsequently hybridized as described in (17).
-
-
-
-
17
-
-
1842292458
-
-
note
-
For in situ hybridization, digoxigenin-or biotin-labeled DNA probes were coprecipitated with herring sperm DNA (10 mg/ml) and yeast tRNA (10 mg/ml), respectively, and resuspended in HB. After prehybridization, probes in HB were transferred to the slide, sealed with a plastic frame (Geneframe, Hybaid, Teddington, UK), and denatured simultaneously with the cellular DNA on an in situ block (Hybaid) at 95°C for 5 min and hybridized at 37°C for 16 hours. Washing was done in 50% formamide, 2× standard saline citrate (SSC) for 30 min at 37°C, 10 min in 2× SSC at 50°C, 60 min in 0.2× SSC at 50°C, and 10 min in 4× SSC at 20°C. For detection of digoxigenin-labeled probes, cells were then incubated with a sheep anti-digoxigenin Fab fragment (Boehringer Mannheim) in 100 mM maleic acid, 150 mM NaCl, 1% blocking reagent (Boehringer) (MEB) for 1 hour at 20°C, washed in tris-buffered saline plus 0.1% Tween-20, and incubated with an FITC-conjugated antibody to sheep immunoglobulin G (Jackson Laboratory) for 45 min in MEB. For detection of biotinylated DNA, cells were incubated with a streptavidin-CY3 conjugate (Sigma) for 1 hour at 20°C in MEB. After washing as above, cells were mounted in Vectashield (Vector Laboratories) containing 4′,6′-diamidino-2-phenylindole (DAPI; 0.1 μg/ml). Cells were analyzed on a Leitz DMRB microscope, and images were captured by means of a slow-scan cooled charge-coupled device camera (Photometrics) and IPLab software (Sigma Analytics). Images were deconvoluted with HazeBuster software (VayTek) and pseudocolored and merged in Adobe Photoshop on a Macintosh 9500/132 computer.
-
-
-
-
18
-
-
1842408052
-
-
note
-
The minichromosomal 177-bp repeat element was amplified by polymerase chain reaction (PCR) with the primers (5′-GCGAATTCTAAATGGTTCTTATACGAATG) and (5′-TACGAAGCTTAACACTAAAGAACAGCGTTG) (6, 7). The ribosomal 5S DMA was amplified with the primers (5′-GCGAATTCAATCTATGCCAATC) and (5′-TACGAAGCTTCTGATCGCCGTACTAACGAG) (20). Purified PCR products were labeled by PCR with either digoxigenin (ribosomal probe) or biotin (minichromosomal probe) with the same sets of primers and a 1:2 ratio of digoxigenin or biotin-dUTP and dTTP. Labeled PCR products were purified on Qiagen spin columns.
-
-
-
-
21
-
-
1842297849
-
-
personal communication
-
S. Melville, personal communication.
-
-
-
Melville, S.1
-
22
-
-
0029029115
-
-
M. Burri et al., Biol. Cell 83, 23 (1995).
-
(1995)
Biol. Cell
, vol.83
, pp. 23
-
-
Burri, M.1
-
24
-
-
1842411580
-
-
unpublished results; K. Ersfeld and K. Gull, unpublished results
-
I. Alieva, I. Vorobjev, E. Ogbadoyi, K. Ersfeld, K. Gull, unpublished results; K. Ersfeld and K. Gull, unpublished results.
-
-
-
Alieva, I.1
Vorobjev, I.2
Ogbadoyi, E.3
Ersfeld, K.4
Gull, K.5
-
25
-
-
0028935746
-
-
D. R. Robinson, T. Sherwin, A. Ploubidou, E. H. Byard, K. Gull, J. Cell Biol. 128, 1163 (1995).
-
(1995)
J. Cell Biol.
, vol.128
, pp. 1163
-
-
Robinson, D.R.1
Sherwin, T.2
Ploubidou, A.3
Byard, E.H.4
Gull, K.5
-
26
-
-
1842371173
-
-
note
-
We thank members of our lab for discussion. DNA probes of the tubulin locus were obtained from S. Melville, University of Cambridge. The work in our lab was supported by a Wellcome Trust Programme and Equipment Grant. K.E. is supported by a Wellcome Trust Postdoctoral Fellowship.
-
-
-
|