Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli

• Published in: Chromosoma 1991-09, Vol.100 (8), p.519-523
• Main Authors: Dvorkin, N, Clark, M.W, Hamkalo, B.A
• Format: Article
• Language: English
• Subjects: Base Sequence; Biological and medical sciences; cell nucleolus; Cell Nucleolus - chemistry; Cell Nucleolus - ultrastructure; cell ultrastructure; Cytogenetics; DNA probes; DNA, Fungal - analysis; DNA, Ribosomal - analysis; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; immunocytochemistry; Immunohistochemistry; in situ hybridization; Microscopy, Electron; Nucleic Acid Hybridization; repetitive sequences; ribosomal DNA; ribosomal RNA; RNA, Fungal - analysis; RNA, Ribosomal - analysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - ultrastructure; small nuclear RNA; Thallophyta, bryophyta; Vegetals and fungi
• ISSN: 0009-5915; 1432-0886
• DOI: 10.1007/BF00352202

The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100-200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rRNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolar-associated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.