Replication kinetics of Chinese hamster chromosomes as revealed by bivariate flow karyotyping

In this paper we show that the replication kinetics of individual chromosomes can be estimated by bivariate flow cytometric analysis of chromosomes isolated from 5‐bromo‐2′‐deoxyuridine (BrdUrd) treated cells. To study the timing of replication of chromosomes, Chinese hamster M3–1 cells were grown for different times in BrdUrd containing medium. Colcemid was added during the last 90 minutes of each BrdUrd labeling period. Chromosomes were isolated from the Colcemid blocked mitotic cells and stained with Hoechst 33258 (HO) and chromomycin A3 (CA3). Bivariate HO‐CA3 flow karyotypes were measured in a dual beam flow cytometer. Labeling cells with BrdUrd for 2, 2 1/2, 3, 3 1/2, and 4 hours before chromosome isolation resulted in progressive quenching of the HO fluorescence of chromosomes Y and 10, 11, M2 while the HO fluorescence of all other chromosomes was either not quenched or was only slightly quenched. The CA3 fluorescence of all chromosomes was slightly enhanced by BrdUrd treatment. A quantitative evaluation of the quenching of HO fluorescence suggested that: a) greater than 50% of HO binding DNA of chromosomes 10, 11, M2 is synthesized in the last quarter of S phase; b) the Y chromosome synthesizes more than 50% of HO binding DNA in the last tenth of S phase making the Y chromosome the latest replicating M3‐1 chromosome. All other chromosomes have synthesized greater than 50% of their HO binding DNA before the last third of S phase.