DNA REPLICATION INITIATION SITES IN MAMMALIAN CELLS

我们目前的研究目标是确定哺乳动物细胞中的DNA复制起始位点，特别关注小鼠免疫球蛋白重链基因（Igh）位点。我们的研究发现，复制与表达的时间组织发生了巨大变化。我们认为这些差异是由于利用不同的复制起始位点。我们计划确定细胞中表达Igh基因座的复制起始位点的位置，并将其与不表达该基因座的位点进行比较。我们已经证明，在小鼠红细胞白血病细胞系中，当其转录失活时，Igh位点被细分为早期和晚期复制域。复制开始于一个早期复制域，在恒定区基因下游的55kb区域内。单个复制分叉完成了从早期复制域到后期复制域的400 kb的转换。这个复制分叉似乎起源于一群早激活复制子中的最后一个，并继续到一群晚激活复制子中的第一个。Igh位点可以代表哺乳动物基因组中存在的许多其他类似过渡区域的范例。我们现在计划确定我们已经确定的起始区域是否包含序列特定的复制起始位点。在b前细胞系中，转录活跃的Igh基因座在S期早期不再存在过渡区，整个基因座在很短的时间间隔内复制。我们已经获得的证据表明，当Igh基因座表达时，至少有一个额外的，前b细胞特异性的起源在Igh- c基因座中被激活。我们将确定起始发生在表达的Igh位点的哪个位置。这将是在哺乳动物细胞中发现的第一个组织特异性复制起源。除了我们一直使用的强大技术外，我们还在开发一种新的方法，结合DNA纤维FISH来识别DNA分子上的特定标记位点和免疫荧光来确定相对于这些标记位置的复制起始位置。到目前为止，起源识别复合体（ORC）尚未涉及任何哺乳动物复制子的起始。鉴定Igh基因座上的复制起始位点将使我们能够确定ORC是否与这些位点结合。

The goal of our current research program is to identify DNA replication initiation sites in mammalian cells with a particular focus on the murine immunoglobulin heavy chain gene (Igh) locus. Our studies identified dramatic changes in the temporal organization of replication with expression. We have suggested that these differences are due to the utilization of different replication initiation sites. We plan to determine the location of replication initiation sites in cells in which the Igh locus is expressed and compare them to sites in which the locus is not expressed. We have demonstrated that the Igh locus, when it is transcriptionally inactive in a murine erythroleukemia cell line, is subdivided into early and late replicated domains. Replication initiates in an early replicated domain, within a 55 kb region downstream of the constant region genes. A single replication fork accomplishes a 400 kb transition from the early replicating to the late replicating domains. This replication fork appears to originate from the last in a cluster of early-activated replicons and proceeds to the first in a cluster of late-activated replicons. The Igh locus could represent a paradigm for many other similar transition regions that are present in mammalian genomes. We now plan to determine whether the initiation region we have identified contains sequence specific replication initiation sites. In the transcriptionally active Igh locus in pre-B cell lines, the transition region is no longer present and the entire locus replicates early in S phase within a narrow interval. We have obtained evidence that at least one additional, pre-B cell-specific, origin is activated in the Igh-C locus when the Igh locus is expressed. We will determine where initiation occurs in the expressed Igh locus. This would be the first tissue specific replication origin(s) identified in mammalian cells. In addition to the powerful techniques we have been using, we are also developing a new approach that combines DNA fiber FISH to identify particular marker sites on DNA molecules and immunofluorescence to determine where replication initiates relative to these marker positions. Thus far, the origin recognition complex (ORC) has not been implicated in the initiation of any mammalian replicon. Identification of replication initiation sites in the Igh locus would allow us to determine whether ORC binds to these sites.