Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads.
The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific sequence on the rRNA nascent transcript was labeled in a pattern consistent with its location; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was systematically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simultaneously maintaining sufficient levels of specific labeling.
O'Reilly
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MM
Department of Biology, University of Virginia, Charlottesville 22903.
French
S L
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Sikes
M L
ML
Miller
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OL
D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry; D002843:Chromatin / Q000648:ultrastructure*; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D009673:Notophthalmus viridescens; D009865:Oocytes / Q000737:chemistry; D011107:Polylysine; D011485:Protein Binding; D018075:RNA, Complementary; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014982:Xenopus laevis
D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018075:RNA, Complementary; D012337:RNA, Ribosomal, 18S; D016601:RNA-Binding Proteins; D011107:Polylysine