Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy

Abstract

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.

Holocentric Chromosomes of <i>Luzula elegans</i> Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region

“…Rather, a 45S rDNA-containing chromatin protrusion might mimic the morphological end of the chromosome. However, no distinct structure associated with terminal NORs was observed [Schroeder-Reiter et al, 2006]. A centromere-free distal end of holocentric chromosomes was also demonstrated for C. elegans , L. nivea [Nagaki et al, 2005], and the insect Oncopeltus fasciatus [Comings and Okada, 1972].…”

Morphological aspects ofPassiflora edulisf.flavicarpachromosomes using acridine orange banding and rDNA-FISH tools

“…As shown by SUMNER (2003), the 45S unit includes the 18S sequence, since the NORs correspond to the sequences of repeated DNA encoding for 18S, 5.8S and 26S rRNAs (BESEN-DORFER et al 2002;SCHROEDER-REITER et al 2006) and, cytologically, these regions are localized in SC in metaphasic chromosomes (BRASILEIRO-VI- DAL et al 2003).…”

Current SEM techniques for de‐ and re‐construction of centromeres to determine 3D CENH3 distribution in barley mitotic chromosomes

“…For three‐dimensional structural investigations, field emission scanning electron microscopy (FESEM) has been successfully applied to visualize chromatin (sub)structures from the elementary fibril, chromomeres and chromosomes in all stages of the cell cycle in mitosis and meiosis (Martin et al , 1994; Wanner & Formanek, 2000; Zoller et al , 2004; Wanner et al , 2005). Analytical SEM techniques, such as energy dispersive X‐ray analysis, artificial decondensation, metal contrasting and immunogold labelling enable high‐resolution investigation of the distribution of DNA, specific DNA sequences and specific proteins in a structural context (Wanner & Formanek, 1995; Wanner & Formanek, 2000; Schroeder‐Reiter et al , 2003; Schroeder‐Reiter et al , 2006). However, until recently, high‐resolution SEM investigations on chromosomes were performed by applying voltages between 5 kV and 30 kV, requiring conductive coating of specimens to prevent interfering charging effects.…”