Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence

Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-of-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.