Scanning electron microscopic detection of nuclear structures involved in DNA replication.

Abstract

In order to evaluate at the ultrastructural level the three dimensional chromatin arrangement during interphase and particularly during the S phase, the immunogold detection of Bromodeoxyuridine (BrdU), as a marker of DNA synthesis, was performed in human HeLa, HL60, and in murine Friend leukemia cells (FLC). Field emission in lens scanning electron microscopy analysis of ultrathin cryosections revealed the presence of a regular three-dimensional network of fibers in dispersed chromatin. This spatial architecture was apparently constituted mainly of 10 nm filaments organized in loops of about 80-100 nm. Nodal points and the overlapping of such coils appeared as thicker 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 analyzed. The DNase treatment of the samples selectively removed the 10 nm class fibers, whereas the BrdU labeling confirmed the presence of newly synthesized DNA organized into chromatin units with a regular arrangement. These data suggest that the 10 nm chromatin fiber likely represents the DNA condensation order at which DNA duplication starts and the main weft of a three dimensional network within the interphase nucleus.