Scanning electron microscopic detection of nuclear structures involved in DNA replication

In order to evaluate at the ultrastructural level the three dimensional chr omatin arrangement during interphase and particularly during the S phase, t he immunogold detection of Bromodeoxyuridine (BrdU), as a marker of DNA syn thesis, was performed in human HeLa, HL 60, and in murine Friend leukemia c ells (FLC). Field emission in lens scanning electron microscopy analysis of ultrathin cryosections revealed the presence of a regular three-dimensiona l network of fibers in dispersed chromatin. This spatial architecture was a pparently constituted mainly of 10 nm filaments organized in loops of about 80-100 nm. Nodal points and the overlapping of such coils appeared as thic ker structures of about 30 nm in diameter. Thin filaments of about 5 nm did not show a regular distribution. This three-dimensional fiber organization seemed quite constant in the dispersed chromatin of all the cell lines ana lyzed. The DNase treatment of the samples selectively removed the 10 nm class fibe rs, whereas the BrdU labeling confirmed the presence of newly synthesized D NA organized into chromatin units with a regular arrangement. These data su ggest that the 10 nm chromatin fiber likely represents the DNA condensation order at which DNA duplication starts and the main weft of a three dimensi onal network within the interphase nucleus.