BrdU-Hoechst-Giemsa analysis of DNA replication in synchronized lymphocyte cultures. Study of human X and Y chromosomes.

Abstract

The combination of a technique of reversible methotrexate (MTX) imposed G1/S block in cultures of human lymphocytes with the BrdU-Hoechst-Giemsa technique permitted the study of DNA replication patterns in individual chromosomes at different intervals of the S phase in a cell cohort with uniform S + G2 duration. The procedure did not increase either the frequency of chromosomal breakage or SCE frequency. The technique applied permitted visualization of the banding pattern in over 90% of mitoses. Examination of mitosis following different times of exposure to BrdU revealed a high degree of synchrony in the progression of the cell cohort examined through the S phase. The presence of two distinct late replication patterns of the allocyclic X chromosome was confirmed in studies on lymphocytes from normal human females by this technique. Interindividual and intercellular differences of the replication pattern have been demonstrated. The replicating patterns from one individual were relatively constant. The analysis of the Y chromosome has revealed marked differences of the termination of replication in individual cells. Euchromatic regions have been shown to complete DNA synthesis first, followed by the distal part of the long arm and, finally, by the region of Yq11/Yq12 junction. Lateral asymmetry was localised at this region.