The beta-major and beta-minor globin genes in murine erythroleukemia cells replicate during the same early interval of the S phase

The time of replication in S phase was determined for the 7.3 kb EcoRI segment containing the beta-major globin gene and the 14 kb EcoRI segment containing the beta-minor globin gene in a murine Friend erythroleukemia virus transformed (MEL) cell line. Cells were obtained from different intervals of S phase by centrifugal elutriation to avoid artifacts of chemical synchronization. Newly synthesized DNA from different parts of S phase were obtained by isolation of 5-bromouracil (BU) labelled DNA from these cells. The BU-DNA synthesized during four different intervals of S was transferred to diazobenzyloxymethyl (DBM) paper and hybridized with a beta-globin cDNA probe. Quantitation showed that both beta-globin segments were replicated in the first quarter of the S phase with no significant difference in their time of replication in this MEL cell line.     

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.     

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 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.     

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.     

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.     

Changes in replication, nuclear location, and expression of the Igh locus after fusion of a pre-B cell line with a T cell line

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3' end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3' to 5' unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) x BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3' Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

Aberrant double replication of segments of chromosomal DNA following DNA synthesis inhibition by cytosine arabinoside.

Using two different cell lines growing logarithmically, it is demonstrated that inhibition of DNA synthesis by cytosine arabinoside leads to a disruption of the sequence of replication of the chromosomal DNA. After release of the inhibition, some DNA synthesis is reinitiated in DNA segments replicated earlier in that S phase, leading to double replication of some DNA segments. This was directly demonstrated by showing that, following removal of the inhibitor, DNA was synthesized off template strands which had themselves been synthesized only 1–2 h before the addition of the inhibitor.     

Estimating the Total Rate of DNA Replication Using Branching Processes

Increasing the knowledge of various cell cycle kinetic parameters, such as the length of the cell cycle and its different phases, is of considerable importance for several purposes including tumor diagnostics and treatment in clinical health care and a deepened understanding of tumor growth mechanisms. Of particular interest as a prognostic factor in different cancer forms is the S phase, during which DNA is replicated. In the present paper, we estimate the DNA replication rate and the S phase length from bromodeoxyuridine-DNA flow cytometry data. The mathematical analysis is based on a branching process model, paired with an assumed gamma distribution for the S phase duration, with which the DNA distribution of S phase cells can be expressed in terms of the DNA replication rate. Flow cytometry data typically contains rather large measurement variations, however, and we employ nonparametric deconvolution to estimate the underlying DNA distribution of S phase cells; an estimate of the DNA replication rate is then provided by this distribution and the mathematical model.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

Molecular analysis of the aberrant replication banding pattern on chromosome 15 in murine T-cell lymphomas

Cytogenetic techniques revealed an altered early replication banding pattern on the distal part of chromosome 15 in some murine T-cell lymphomas. This pattern reverted back to normal replication in somatic cell hybrids that had become non-tumorigenic after fusion of leukemic cells with normal fibroblasts. The altered banding pattern was correlated with malignancy. To investigate the molecular basis of the aberrant pattern in more detail, centrifugal elutriation of cells containing bromodeoxyuridine labeled DNA was used to prepare newly replicated DNA from selected intervals of the S-phase from tumor cells, as well as from hybrid cells with the revertant phenotype. These different DNA fractions were probed for DNA sequences distributed over the distal half of chromosome 15. Only two out of ten chromosome 15 specific genes tested showed a clear change in replication timing between the two different cell lines tested. These two genes were the lymphocyte antigen-6,Ly-6, and the neighboring thyroglobulin gene,Tgn, which replicated at the beginning of S in the tumor cells and later in S in the non-tumorigenic hybrid cells.by J.A. Huberman     

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.