alpha-Globulin sequences are located in a region of early-replicating DNA in murine 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 murine erythroleukemia cell line transformed by Friend virus. Cells grown for short times in the presence of BrdUrd were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases, and fragments containing bromouracil (BrU-DNA) were isolated in density gradients of Cs2SO4. BrU-DNA fractions replicated during selected S-phase intervals were subjected to electrophoresis in agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to an alpha-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one alpha-globin gene demonstrated that the extent of hybridization provides an accurate measurement that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The alpha-globin fragments were detected primarily in the BrU-DNA replicated during early S phase (approximately the first quarter of S). This result was confirmed in other synchrony experiments and by Cot analysis. (Cot is the initial concentration of DNA in mol of nucleotide per liter multiplied by the time in sec.) The temporal replication of mouse satellite sequences, already known from previous studies, was used as an internal control for cell synchrony. To show that the globin sequences were not lost from the cells in late S phase during isolation of the DNA, we quantitated th alpha-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the alpha-globin gene regions in these cells are replicated during early S phase.