The temporal order of replication of murine immunoglobulin heavy chain constant region sequences corresponds to their linear order in the genome

The time of replication during the S phase in a murine erythroleukemia (MEL) cell 1ine was determined for imnunoglobulin heavy chain constant region Cα, Cγ2b and Cμ sequences whose boundaries are defined by EcoRl restriction endonuclease sites (EcoR1 segments). Logarithmically growing cultures of MEL cells with an S phase of about 7.5 hours were pulse labelled with 20 μg/ml of 5-bromodeoxyuridine (BUdR) . The cells were then fractionated by centrifugal elutriationin to 10-12 distinct populations containing cells in diferent stages of the cell cycle. Flow microfluorimetric (FMF) analysis of DNA content, measurements of cell volume and autoradiograph, after ³H-thymidine pulse labelling were used to determine positionin the cell cycle. Fractions were pooled to represent four selected intervals of 5 in which BU-DNA was synthesized for 2.5 hrs or less. Newly replicated DNA which had incorporated BUdR into one strand was isolated, cleaved with EcoRl, and separated on neutral Cs₂ SO₄ gradients. Equal amounts of BU-DNA replicated during these four intervals of S were electrophoresed i n 0.8% agarose gels, transferred to diazotized aminobenzyloxflethyl paper and hybridized with ₃₂P probes containing the Cα, Cγ2b and Cμ qenes and flanking sequences. The relative amounts of segments replicated were assessed by quantitation of the appropriate bands on the autoradiograms by microdensitometry. The results indicate that the 2.8 kb Cα, 6.6 kb Cγ2b and 12 kb Cμ EcoRl segments in these MEL cells replicated during defined intervals of the first half of the S phase. The order of replication of these EcoRl segments as the cells proceeded through S was Cα, Cγ2b, Cμ, corresponding to the linear order of the genes determined by restriction endonuclease mapping.