We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three
triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative
triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical
stain to measure X-linked
glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2)
cellulose acetate electrophoresis of G6PD activity in
cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of
47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in
BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the
triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.