Abstract
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 nucleolarassociated 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.
Similar content being viewed by others
Abbreviations
- snRNA:
-
small nuclear RNA
- AAF:
-
N-acetoxy-2-acetyl-aminofluorence
References
Byers, B (1981) Cytology of the yeast life cycle. In: Molecular biology of the yeast Saccharomyces: Life cycle and inheritance. Strathern, JN, Jones, EW, Broach, JR (eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 59–96
Clark, MW (1991) Immunogold labelling of yeast ultrathin sections. Methods Enzymol 194:608–626
Clark, MW, Abelson, J (1987) The subnuclear localization of tRNA ligase in yeast. J Cell Biol 105:1515–1526
Clark, MW, Yip, MLR, Campbell, J, Abelson, J (1990) SSB-1 of the yeast, Saccharomyces cerevisiae, is a nucleolar-specific silver-binding protein that is associated with the snR10 and snR11, small nuclear RNAs. J Cell Biol 111:1741–1752
Hirano, T, Konoha, G, Toda, T, Yanagida, M (1989) Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus. J Cell Biol 108:243–253
Hughes, JMX, Konings, DAM, Cesareni, G (1987) The yeast homologue of U3 snRNA. EMBO J 6:2145–2155
Hurt, EC, McDowell, A, Schimmang, T (1988) Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae. Eur J Cell Biol 46:554–563
Landegent, JE, Jansen in de wal, N, Baan, RA, Hoeijmakers, JHJ, van derPloeg, M (1984) 2-acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp Cell Res 153:61–72
Miller, OLJr, Beatty, BR (1969) Visualization of nucleolar genes. Science 164:955–957
Narayanswami, S, Lundgren, K, Hamkalo, BA (1989) Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy. Scanning Microsc [Suppl] 3:65–76
Parker, KA, Bruzik, JP, Steitz, JA (1988) An in vitro interaction between the human U3 snRNP and 28S rRNA sequence near the alpha-sarcin site. Nucleic Acids Res 16:10493–10503
Petes, TD, Botstein, D (1977) Simple mendelian inheritance of the reiterated ribosomal DNA of yeast. Proc Natl Acad Sci USA 74:5091–5095
Petes, TD, Hereford, LM, Skryabin, KC (1978) Characterization of two types of yeast ribosomal DNA genes. J Bacteriol 134:295–305
Potashkin, JA, Derby, RJ, Spector, DL (1990) Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus. Mol Cell Biol 10:3524–3534
Rattner, JB, Saunders, C, Davie, JR, Hamkalo, BA (1982) Ultrastructural organization of yeast chromatin. J Cell Biol 92:217–222
Robinow, CF, Marak, J (1966) A fiber apparatus in the nucleus of the yeast cell. J Cell Biol 29:129–132
Schimmang, T, Tollervey, D, Kern, H, Frank, R, Hurt, EC (1989) A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J 8:4015–4024
Sillevis-Smitt, WW, Vlak, JM, Molenaar, I, Rozijn, TH (1983) Nucleolar function of the dense crescent in the yeast nucleus. Exp Cell Res 80:313–321
Szent-Gyorgi, C, Finkelstein, DB, Garrard, WT (1987) Sharp boundaries demarcate the chromatin structure of a yeast heat-shock gene. J Mol Biol 193:71–80
Tollervey, D, Mattaj, TW (1987) Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J 6:469–476
Tyc, K, Steitz, JA (1989) U3, U8, and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J 8:3113–3119
Author information
Authors and Affiliations
Additional information
by M.L. Pardue
Rights and permissions
About this article
Cite this article
Dvorkin, N., Clark, M.W. & Hamkalo, B.A. Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli. Chromosoma 100, 519–523 (1991). https://doi.org/10.1007/BF00352202
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00352202