Replication and subnuclear localization of the murine immunoglobulin heavy chain gene locus
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Two dramatically different replication programs have been observed for the murine immunoglobulin heavy chain (Igh) 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 the immature B WEHI-231 and the plasma cell line S107, there is a temporal transition region (∼500 kb) that links an early replication domain located downstream of the Igh locus and a late replication domain within the variable region genes. Associated with the contrasting replication programs are differences in the subnuclear location of the Igh locus. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery. However, when there is a temporal transition region, the entire locus is located near the nuclear periphery. Thus, the change in subnuclear localization could play a role in regulating the replication of the Igh locus.;Previous studies have shown that replication forks progressed in a 3 ' to 5' direction through the transition region in MEL cells, while replication forks progressed in both directions through the constant region genes in pre B cell lines. Here, I have used a RAG 2-/- cell line to show that bidirectional fork movement is independent of VDJ recombination. Moreover, I found that replication forks progress in a single 3' to 5' direction in a pre-B and T cell fusion cell line in which the B cell program is inactive. Therefore, pre-B cell specific factors may be important for activation of a pre-B cell specific origin. I have also demonstrated that forks progress in a 3' to 5' direction within the examined constant region genes in WEHI-231. The differences in replication fork direction suggest a molecular mechanism for the different replication programs of the Igh-C region.;Using neutral/alkaline two-dimensional gels, I have localized a replication origin within a 3 kb segment for the temporal transition region in the NOEL cell line. Using the newly developed approach Single Molecule Analysis of Replicated DNA (SMARD) to study DNA replication of the Igh-C locus in MEL cells, I demonstrated that SMARD could be a powerful technique for the study of the Igh locus in primary B lineage cells.
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