Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase

Abstract

We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

“…Although the gold density over the fibrillar component is lower, it is highly significant relative to those compartments without DNA, such as the cytoplasm and the resin. We conclude from this experiment that DNA is certainly present within the NORs (i.e., fibrillar structures identified within chromosomes) and, because the gold density is directly linked to the compaction of DNA (Thiry et al, 1993), that this DNA is around ten times less condensed than DNA in compact chromatin.…”

“…By using osmium ammine complex (Feulgen-like reaction), it was demonstrated that a loose network of nonnucleosomal DNA fibers (a characteristic of rDNA genes) was exclusively colocalized with silver deposits at the level of silver-stained metaphase chromosomes (HernandezVerdun and Derenzini, 1983). Moreover, by using the TdT method (Thiry et al, 1993), we unequivocally localized DNA within chromosomic fibrillar domains previ- These views show the hypothetical levels of folding of the r-chromatin fiber during prophase and metaphase. In this 3D model, the r-chromatin fiber (i.e., rDNA genes associated with Ag-NOR proteins) constitute one fiber 80 nm in diameter (in yellow) emanating from an AT-rich axis 250 nm in diameter (in red) of one chromatid.…”

“…DNA localization within NORs was performed on Ehrlich ascites tumor cells grown as previously described and blocked in metaphase with Colcemid (Thiry et al, 1993). After fixation and dehydration, cells were acetylated to better differentiate chromatin from the NORs on ultrathin sections (Ploton et al, 1987).…”

“…After fixation and dehydration, cells were acetylated to better differentiate chromatin from the NORs on ultrathin sections (Ploton et al, 1987). Ultrastructural localization of DNA was performed on ultrathin sections by using the highly specific TdT method (terminal deoxynucleotidyltransferase immunogold method; Thiry et al, 1993). After counterstaining with uranyl and lead, gold particles 10 nm in diameter could be easily localized relative to the cellular components; more particularly, chromatin appears as compact areas with a high contrast and NORs are easily identified as fibrillar regions with a low contrast (Ploton et al, 1987).…”

“…We chose the TdT method (Thiry et al, 1993;Thiry and Goessens, 1996) (A-C) Three serial tangential sections parallel to the long axis of one NOR-bearing chromosome. After uranyl and lead counterstaining, chromatin (arrow 1) appears with a high contrast to electrons, whereas the fibrillar component of one NOR shows a lower contrast (arrow 2).…”