The time of replication during the S phase in a murine erythroleukemia (MEL) cell line was determined for immunoglobulin heavy chain constant region C alpha, C gamma 2b and C mu sequences whose boundaries are defined by EcoR1 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 micrograms/ml of 5-bromodeoxyuridine (BUdR). The cells were then fractionated by centrifugal elutriation into 10-12 distinct populations containing cells in different stages of the cell cycle. Flow microfluorimetric (FMF) analysis of DNA content, measurements of cell volume and autoradiography after 3H-thymidine pulse labelling were used to determine position in the cell cycle. Fractions were pooled to represent four selected intervals of S 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 EcoR1, and separated on neutral Cs2S04 gradients. Equal amounts of BU-DNA replicated during these four intervals of S were electrophoresed in 0.8% agarose gels, transferred to diazotized aminobenzyloxymethyl paper and hybridized with 32p probes containing the C alpha, C gamma 2b and C mu genes 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 C alpha, 6.6 kb C gamma 2b and 12 kb C mu EcoR1 segments in these MEL cells replicated during defined intervals of the first half of the S phase. The order of replication of these EcoR1 segments as the cells proceeded through S was C alpha, C gamma 2b, C mu, corresponding to the linear order of the genes determined by restriction endonuclease mapping.

The time of replication in the S phase of regions of the mouse genome including the a-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 a-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one a-globin gene demonstrated that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The a-globin fragments were detected primarily in the BrU-DNA 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 the a-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the at-globin gene regions in these cells are replicated during early S phase.DNA sequences in eukaryotes appear to be replicated in a defined temporal order that is maintained from one cell cycle to the next (reviewed in ref. 1). However, little is known about the mechanism by which this temporal control of replication is achieved. There is little information concerning the temporal replication of nonreiterated sequences, nor is it known if the order of replication differs in different cell types.The globin genes have been studied extensively in many organisms (2-5). Several a-globin-like genes in mammalian genomes and their close linkage in humans have been described (2). In Southern transfer experiments with mouse DNAs cleaved by the EcoRI restriction enzyme, which does not cut within these genes, three prominent bands are detected upon hybridization to a probe homologous to a-globin mRNA (3). The band at 10 kilobases (kb) is a doublet, containing the two genes encoding the adult a-globin chains: the a-i gene, on a 9.7-kb fragment (4), and the a-2 gene, on an 11.8-kb fragment (5). We refer to the sequences on these fragments as the a-globin gene regions. Two smaller fragments are also detected that contain a-globin sequences not expressed in adult mice (5).We present evidence here that in a Friend erythroleukemia cell (Friend cell) line, a-globin gene sequences are located in a region of DNA that replicates during early S phase. Synchronous populations of Friend cells were isolated by centrifugal elutriation after growth for short intervals in the presence of BrdUrd. Samples of bromouracil-substituted DNA (BrU-DNA), replicated during selected S-phase intervals, were cleaved to completion wit...

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