DNase I-sensitive sites within the nuclear architecture visualized by immunoelectron microscopy.
Nick-translation using mild digestion with DNase I allows preferential labeling of actively transcribing or potentially active genes, as compared with inactive genes. We have adapted this method to the level of electron microscopy to see the DNase I-sensitive regions in situ in Ehrlich tumor cells. In interphase cells treated with very low concentrations of DNase I, labeled sequences are found at the borders and in the close vicinity of condensed chromatin blocks. Labeling of condensed areas of chromatin requires higher DNase I concentrations and longer incubation in the nick-translation medium. In the nucleolus, the first sites to be nick-translated are the fibrillar centers and the interstices surrounding them, whereas the dense fibrillar component never contains labeled sequences. When cells are pretreated with actinomycin D, only a few perinucleolar clumps of condensed chromatin are labeled under the same conditions. This method provides a new tool for studying the functional organization of chromatin within a cell. The precise location of nick-translated sites in nucleolar components observed could change classical views concerning the functional organization of the nucleolus.
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Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, Belgium.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000473:pathology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D003609:Dactinomycin / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005821:Genetic Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D012335:RNA, Ribosomal / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012335:RNA, Ribosomal; D003609:Dactinomycin; D003850:Deoxyribonuclease I