Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy
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An atypical peg-like terminal constriction (“peg”) on metaphase chromosomes of the plant genus Oziroë could be identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH). Using high-resolution 3D analytical SEM, the architecture and DNA distribution of the peg-like NOR were characterized as typical for chromosomes, albeit with significantly smaller chromomeres. ISH procedure was improved for SEM concerning signal localization, labeling efficiency, and structural preservation, allowing 3D SEM analysis of the peg-like NOR structure and rDNA distribution for the first time. It could be shown that implementation of FluoroNanogold markers is an attractive tool that allows efficient immunodection in both LM and SEM. A model is proposed for the peg structure and its mode of condensation.
KeywordsNucleolus Organize Region Parallel Fiber Label Efficiency Secondary Constriction Saline Sodium Citrate
The authors thank Katrin Kumke, Sabine Steiner, and Emilie Vosyka for excellent technical assistance, Dr. Helmut Formanek for supplying the Pt blue compound and for support in researching molecular sizes, and Renate Reichinger-Bock for artwork.
- Allen TD, Jack EM, Harrison CJ (eds) (1988) The three dimensional structure of human metaphase chromosomes determined by scanning electron microscopy. Chromosomes and Chromatids. CRC, FloridaGoogle Scholar
- Castilho A, Heslop-Harrison JS (1995) Physical mapping of 5S and 18S–25S rDNA and repetitive DNA sequences in Aegilops umbellulata. Genome 38:91–96Google Scholar
- Guaglianone ER, Arroyo-Leuenberger S (2002) The South American genus Oziroë (Hyacinthaceae–Oziroeoideae). Darwiniana 40(1–4):61–76Google Scholar
- Lehfer H, Wanner G, Herrmann RG (1991) Physical mapping of DNA sequences on plant chromosomes by light microscopy and high resolution scanning electron microscopy. Plant Mol Biol 2:277–284Google Scholar
- Martin R, Busch W, Herrmann RG, Wanner G (1995) In situ hybridization and signal detection by high resolution scanning electron microscopy. In: Brandham PE, Bennet MD (eds) Kew Chromosome Conference IV. Royal Botanic Gardens, Kew, England, pp 159–166Google Scholar
- Pontes O, Lawrence RJ, Neves N, Silva M, Lee J-H, Chen J, Viegas W, Pikaard CS (2003) Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in Arabidopsis. Proc Natl Acad Sci U S A 100(20):11418–11423PubMedCrossRefGoogle Scholar
- Speta F (1998) Systematische Analyse der Gattung Scilla L. s.l. (Hyacinthaceae). Phyton (Horn) 38:1–141Google Scholar
- Yakura K, Tanifuji S (1983) Molecular cloning and restriction analysis of EcoRI-fragments of Vicia faba rDNA. Plant Cell Physiol 24:1327–1330Google Scholar
- Zoller JF (2003) Hochauflösende Strukturanalyse pflanzlicher Chromosomen in Mitose und Meiose. Dissertation der Fakultät für Biologie. Ludwig-Maximilians-Universität München, Munich, Germany (248 pages)Google Scholar