Rate of Replication of the Murine Immunoglobulin Heavy-Chain Locus: Evidence that the Region is Part of a Single Replicon

Abstract

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Gene replication in the presence of aphidicolin

“…Accordingly, the replication of the c-mos gene, which is seen 1 h after the c-Ha-ras gene in the absence of APC, was detected approximately 16 h later in the presence of APC (Figure 3, Table I). Although early-replicating and late-replicating genes can be distinguished during replication in the absence of APC (Brown et al, 1987;Doggett et al, 1988), it is difficult to determine subtle differences in the timing of replication and the correct order of replication of early-replicating genes. However, when replication is attenuated by APC, it is possible to clearly define the temporal order of gene replication.…”

“…Ac- cordingly, studies of replication in the presence of APC may have applications for understanding the dynamics of events at ori and termination regions during replication of mammalian chromosomes. For example, putative termination and origin regions for DNA replication in mammalian cells have been described (Epner et al, 1988;Brown et al, 1987).…”