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Volumn 276, Issue 5312, 1997, Pages 611-614

Partitioning of large and minichromosomes in Trypanosoma brucei

Author keywords

[No Author keywords available]

Indexed keywords

GLYCOPROTEIN;

EID: 0030978125     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5312.611     Document Type: Article
Times cited : (84)

References (26)
  • 16
    • 1842375356 scopus 로고    scopus 로고
    • 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 scopus 로고    scopus 로고
    • 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 scopus 로고    scopus 로고
    • 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 scopus 로고    scopus 로고
    • personal communication
    • S. Melville, personal communication.
    • Melville, S.1
  • 22
  • 26
    • 1842371173 scopus 로고    scopus 로고
    • 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.


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