Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Abstract

Electron microscopy of unstained BrdU-substituted chromosomes treated with 1.0 M NaH₂PO₄ at high pH and high temperature has demonstrated that there is a structural basis for the light microscopic observation of differentially Giemsa-stained unifilarly and bifilarly BrdU-substituted chromatids and the appearance of chromosome dots. At progressively higher treatment temperatures, sequential structural changes occurred in the chromosomes. After treatment with NaH₂PO₄ at 70–80° C, unifilarly BrdU-substituted chromatids were much more electron opaque than bifilarly substituted chromatids, and the overall data suggest that this difference in electron opacity is a result of the preferential extraction of chromosomal DNA from the bifilarly BrdU-substituted chromatids. NaH₂PO₄ treatment of the BrdU-substituted chromosomes at 80–90° ° C resulted in the formation of highly electron opaque spots (dots) on one or both chromatids. Dots first appeared on the electron lucent bifilarly BrdU-substituted chromatid, indicating that the chromatin with the greatest substitution of BrdU in its DNA is most susceptible to dot formation. At a slightly higher temperature, dots also appeared on the unifilarly BrdU-substituted chromatid concomitant with a disappearance of the electron opacity characterizing this chromatid at the lower treatment temperature. The dots may be formed by an extreme reorganization of residual chromatin or by some kind of interaction or reaction between the chromatin and the salts in the incubation medium. G-band regions may serve as focal points for dot formation.