Characterization of Giemsa dark- and light-band DNA

Abstract

Using synchronized cultures of V79-8 Chinese hamster lung fibroblasts, we either alternately labeled early- and late-replicating DNA, or substituted one of these with bromodeoxyuridine to separate them in CsCl density gradients or to identify the bromodeoxyuridine-containing chromosome bands by fluorescence microscopy. The Giemsa light R bands were shown to replicate in the first half of S phase, and the dark G bands were shown to replicate in the last half of S phase. S phase was bimodal, with a distinct pause in the rate of DNA synthesis that separated the period of R-band DNA synthesis from that of G-band DNA synthesis. G-band DNA was found to be 3.2% richer in AT than R-band DNA. Surprisingly, G- and R-band DNA appeared equally transcriptionally active in that alternate labels in chromatin were digested with the same kinetics by DNAase I, and in reassociation experiments, total poly(A)+ RNA drove nick-translated G- and R-band DNA probes similarly. G- and R-band DNA also reassociated with identical kinetics, demonstrating that they contain equal proportions of all kinetic-complexity classes of sequences.