Replication of Mammalian Chromosomes

描述(申请人提供)：dhfr起源由22个低效起始点组成，散布在可转换转录的dhfr和2BE2121基因之间的55kb间隔区。然而，有些站点(Ori-？和Ori-？)显然更受欢迎。我们已经证明，在从DHFR起源的最活跃的起始位点，甚至包括90%的活性位点的中心40kb核心的基因缺失中，并不抑制间隔区其余部分的起始。事实上，启动频率实际上增加了。因此，主复制子并不驻留在原点本身。然而，活性的dhfr启动子是获得最大起始活性所必需的，如果没有活性启动子，复制就会在现已失活的基因中启动。此外，该基因的3‘端处理信号通过通读转录防止了起始点的失活。我们认为哺乳动物基因组以1kb的间隔点缀着简并复制子，其活性受局部转录和染色质结构的调节。具体目标是：1)确定被认为代表真正复制子的人层蛋白B2和珠蛋白起始点是否本质上比DHFR起始区中最活跃的位点更活跃；Ori-1将与不匹配的loxP位点侧翼交换，并与两个人类起始点交换，或与在Loco中不能有效启动的片段交换；然后将量化每个起始点的活性；也将在人类细胞中将lamin B2和？-珠蛋白与位于起始区的片段进行比较；2)确定Ori-和Ori-？由于它们相对于本地转录单位的位置，它们在间隔区中比其他位点更活跃；通过Roko方法，Ori-？将在间隔区中定位不同，并评估对起始点效率的影响；3)确定DHFR和较弱的2BE2121启动子是否在起始点激活中发挥必要但多余的作用；两个启动子都将被删除，并对起始点活性的影响进行评估；此外，将在DHFR启动子下游放置一个强烈的转录停止信号，以确定起始点刺激是否需要转录分叉运动(而不是启动子本身)；4)确定当基因间隔区被截断时启动效率增加的原因，并准备一个“超级”起始点；间隔区的长度将被缩短以确定最佳大小，并将在dhfr和2BE2121基因下游放置一个强大的转录终止子；5)确定当Ori-1因5‘和3’调控元件缺失而失活或被上述策略之一激活时所发生的表观遗传学变化；将利用芯片技术来确定起始蛋白和修饰的组蛋白在AIMS 1-4中阐述的变体中作为细胞周期函数的分布；这些研究将指出限制起始效率的因素，并将检验通过间隔区转录移除前RCS，而启动子缺失导致无法加载它们的预测。

DESCRIPTION (provided by applicant): The DHFR origin consists of a zone of >22 inefficient initiation sites scattered throughout the 55 kb spacer between the convergently-transcribed DHFR and 2BE2121 genes. However, some sites (ori-¿ and ori-?) are clearly preferred. We have shown that in loco deletion of the most active initiation sites from the DHFR origin, or even the central 40-kb core that encompasses >90% of active sites, does not suppress initiation in the remainder of the spacer. In fact, initiation frequency actually increases. Thus, master replicators do not reside in the origin itself. However, an active DHFR promoter is required for maximum origin activity and, in its absence, replication initiates in the now-inactive gene. Furthermore, the 3' processing signals of the gene prevent inactivation of the origin by read-through transcription. We propose that mammalian genomes are peppered at intervals of <1 kb with degenerate replicators whose activities are modulated by local transcription and chromatin architecture. Specific aims are: 1) to determine whether the human lamin B2 and ¿-globin origins, which are thought to represent true replicators, are intrinsically more active than the most active sites in the DHFR initiation zone; ori-¿ will be flanked with mismatched LoxP sites and exchanged with the two human origins, or with fragments that do not initiate efficiently in loco; the activities of each will then be quantified; lamin B2 and ¿-globin also will be compared in human cells to fragments residing in initiation zones; 2) to determine whether ori-¿ and ori-? are more active than other sites in the spacer because of their locations vis-¿-vis the local transcription units; ori-¿ will be positioned differently in the spacer by the ROKO approach, and effects on origin efficiency assessed; 3) to determine whether DHFR and the weaker 2BE2121 promoters play essential but redundant roles in origin activation; both promoters will be deleted, and effects on origin activity will be assessed; in addition, a strong transcription stop signal will be placed just downstream from the DHFR promoter to determine whether transcription fork movement (as opposed to the promoter per se) is required for origin stimulation; 4) to determine why the efficiency of initiation increases when the intergenic spacer is truncated, and to prepare a "super" origin; the length of the spacer will be reduced to determine optimal size and a strong transcription terminator will be placed downstream from the DHFR and 2BE2121 genes; 5) to define the epigenetic changes that occur when ori-¿ is inactivated by deletion of 5' and 3' regulatory elements or activated by one of the above strategies; ChIP technology will be utilized to determine the distributions of initiation proteins and modified histones as a function of the cell cycle in variants elaborated in Aims 1-4; these studies will indicate factors that limit origin efficiency, and will test the predictions that transcription through the spacer removes pre-RCs, while promoter deletions result in a failure to load them.