Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells

被引:15
作者
Brown, K [1 ]
机构
[1] Hammersmith Hosp, Imperial Coll, Fac Med, MRC Clin Sci Ctr, London W12 0NN, England
关键词
nuclear organization; immuno-fluorescence in situ hybridization; immunocytochemistry; fluorescence in situ hybridization;
D O I
10.1016/S1046-2023(02)00003-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Recent data support the idea that the mammalian nucleus is organized in a functionally significant way. Immunocytochemistry has revealed the existence of diverse nuclear "bodies" and compartments. Fluorescence in situ hybridization (FISH) lids shown that chromosomes change their spatial relationships during dynamic cell cycle progression and that nuclear organization can change during development and differentiation when patterns of gene expression are established or modified. To determine the relationship between nuclear organization and genome function is an important goal for biology. This article describes an immunoFISH technique, which is a useful tool for investigating the functional organization of the nucleus. It combines immunocytochemistry with FISH to allow associations between proteins, DNA, and RNA to be visualized in a single-step analysis using confocal microscopy. Immunocytochemistry and FISH were thought incompatible since cell preparation flattens nuclei and the harsh DNA denaturation treatment required for FISH destroys proteins. The immunoFISH technique successfully overcomes these problems and can reveal interactions between nuclear components not readily detectable using other experimental approaches. The interactions of single-copy, endogenous loci with nuclear proteins or bodies can be seen, as can spatial compartmentalization of these loci, in cells preserved in three dimensions, representative of the situation in vivo. Allelic differences in transcription can be related to nuclear location and protein interactions of the individual alleles since genes, RNA and proteins can be visualized together. Chromosome behavior can be followed through mitosis to investigate centromere activity or vector segregation efficiency, for example, Visual data obtained using the immunoFISH technique have provided insight into the functional significance of nuclear organization and its role in cell biology. (C) 2002 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:10 / 18
页数:9
相关论文
共 38 条
  • [1] Bickmore W, 1999, METHOD ENZYMOL, V304, P650
  • [2] The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells
    Boyle, S
    Gilchrist, S
    Bridger, JM
    Mahy, NL
    Ellis, JA
    Bickmore, WA
    [J]. HUMAN MOLECULAR GENETICS, 2001, 10 (03) : 211 - 219
  • [3] Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts
    Bridger, JM
    Boyle, S
    Kill, IR
    Bickmore, WA
    [J]. CURRENT BIOLOGY, 2000, 10 (03) : 149 - 152
  • [4] Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division
    Brown, KE
    Baxter, J
    Graf, D
    Merkenschlager, M
    Fisher, AG
    [J]. MOLECULAR CELL, 1999, 3 (02) : 207 - 217
  • [5] Expression of α- and β-globin genes occurs within different nuclear domains in haemopoietic cells
    Brown, KE
    Amoils, S
    Horn, JM
    Buckle, VJ
    Higgs, DR
    Merkenschlager, M
    Fisher, AG
    [J]. NATURE CELL BIOLOGY, 2001, 3 (06) : 602 - 606
  • [6] DISSECTING THE CENTROMERE OF THE HUMAN Y-CHROMOSOME WITH CLONED TELOMERIC DNA
    BROWN, KE
    BARNETT, MA
    BURGTORF, C
    SHAW, P
    BUCKLE, VJ
    BROWN, WRA
    [J]. HUMAN MOLECULAR GENETICS, 1994, 3 (08) : 1227 - 1237
  • [7] Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin
    Brown, KE
    Guest, SS
    Smale, ST
    Hahm, K
    Merkenschlager, M
    Fisher, AG
    [J]. CELL, 1997, 91 (06) : 845 - 854
  • [8] BROWN KE, 1995, THESIS U OXFORD
  • [9] BUCKLE VJ, 1993, FLUORESCENT IN SITU, P59
  • [10] Nuclear compartments and gene regulation
    Cockell, M
    Gasser, SM
    [J]. CURRENT OPINION IN GENETICS & DEVELOPMENT, 1999, 9 (02) : 199 - 205