Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus.
Post-embedding electron microscope in situ hybridization using gold particles as label permits the clear identification of the cellular structures which contain the nucleic acid molecules under study. It has yielded information on the distribution of defined nucleic acid sequences of different origins-cellular or viral, DNA or RNA, single- or double-stranded molecules-which has revolutionized the study of the nucleus. Application of this powerful technique in combination with other refined techniques to studies on transcription and replication of cellular and viral genes has augmented our knowledge of the functional organization of the cell nucleus. One can now ask mechanistically meaningful questions concerning the successive steps of gene replication and expression not only under normal conditions of cell growth, but also when the cellular metabolism is altered by a drug treatment or a viral infection. This chapter aims (a) to present the established methods of post-embedding electron microscope in situ hybridization for localizing, precisely and specifically, a nucleic acid target in its normal environment and (b) to present some contributions of this technique to investigations of the functional compartmentalization of the cell nucleus and to elucidate the cell-virus relationships in infected cells.
Puvion-Dutilleul
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F
Laboratoire Organisation fonctionelle du Noyau de l'UPR 9044 CNRS, Villejuif, France.
Puvion
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E
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure* / Q000821:virology; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007553:Isotope Labeling; D008854:Microscopy, Electron; D012313:RNA / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis
D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA