THE ALPHA-GLOBIN GENES ARE LOCATED IN A REGION OF EARLY-REPLICATING DNA IN FRIEND ERYTHROLEUKEMIA CELLS

Abstract

The time of replication in the S phase of regions of the mouse genome including the (alpha)-globin genes was determined in the Friend virus-transformed murine erythroleukemia cell line. Cells grown for short times in the presence of 5-bromodeoxyuridine (BUdR) were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases and fragments containing bromouracil (BU-DNA) were isolated in density gradients of cesium sulfate. BU-DNA fractions replicated during selected intervals of the S phase were electrophoresed in agarose gels, transferred to diazobenzyloxymethyl paper, and hybridized to an (alpha)-globin probe. Reconstruction experiments using a cloned mouse Eco R1 fragment containing one of the (alpha)-globin genes demonstrated that the extent of hybridization provides an accurate measure of the concentrations of specific fragments in a DNA sample. The (alpha)-globin fragments were detected primarily in the BU-DNA replicated during early S. This result was confirmed in other experiments in which the methods of cell synchrony and of BU-DNA isolation were varied. The temporal replication of mouse satellite DNA sequences, already known from previous studies, was determined as an internal control for the cell synchrony. To show that the globin sequences were not lost from the cells in late S during isolation of the DNA, the (alpha)-globin fragments were quantitated in BU-DNA prepared from a mixture of cells in early and in late S. The results demonstrate that the (alpha)-globin gene regions are replicated during early S in these cells.