DNA REPLICATION INITIATION SITES IN MAMMALIAN CELLS

哺乳动物细胞中的 DNA 复制起始位点

• 批准号: 6342845
• 负责人: CARL L SCHILDKRAUT
• 金额: $ 46.96万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-08 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342845
• 关键词: B lymphocyte; DNA footprinting; DNA replication origin; Epstein Barr virus; cell line; chromatin; fluorescent in situ hybridization; gel electrophoresis; gene mutation; genetic mapping; genetic regulatory element; immunofluorescence technique; immunoglobulin genes; immunoprecipitation; replicon; sequence tagged sites; species difference; transcription factor

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.

我们目前的研究计划的目标是确定在哺乳动物细胞中的DNA复制起始位点，特别是对小鼠免疫球蛋白重链基因（Igh）位点的关注。我们的研究确定了复制与表达的时间组织的戏剧性变化。 我们认为，这些差异是由于利用不同的复制起始位点。 我们计划确定在细胞中的Igh基因座表达的复制起始位点的位置，并将它们与该基因座不表达的位点进行比较。我们已经证明，免疫球蛋白基因座，当它是在小鼠红白血病细胞系转录失活，被细分为早期和晚期复制域。复制起始于恒定区基因下游55 kb区域内的早期复制结构域。 单个复制叉完成从早期复制域到晚期复制域的400 kb过渡。 这个复制叉似乎起源于早期激活的复制子簇中的最后一个，并继续进行到晚期激活的复制子簇中的第一个。 免疫球蛋白基因座可以代表哺乳动物基因组中存在的许多其他类似过渡区的范例。 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异性复制起始位点。在前B细胞系中的转录活性Igh基因座中，过渡区不再存在，并且整个基因座在S期早期在狭窄的间隔内复制。 我们已经获得的证据表明，至少有一个额外的，前B细胞特异性，起源激活IgH-C基因座时，IgH基因座表达。 我们将确定在表达的Igh基因座中起始发生的位置。 这将是在哺乳动物细胞中鉴定的第一个组织特异性复制起点。 除了我们一直在使用的强大技术外，我们还开发了一种新方法，该方法结合DNA纤维FISH来识别DNA分子上的特定标记位点，并结合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，起源识别复合物（ORC）尚未涉及任何哺乳动物复制子的起始。 Igh基因座中复制起始位点的鉴定将使我们能够确定ORC是否与这些位点结合。