Replication of Mammalian Chromosomes

描述（由申请人提供）：DHFR起源由bbbb22个低效起始位点组成，这些起始位点分散在聚集转录的DHFR和2BE2121基因之间的55kb间隔中。然而，有些网站（ori-¿和ori-?）显然是首选。我们已经证明，从DHFR起源中删除最活跃的起始位点，甚至是包含b> 90%活性位点的中央40kb核心，都不会抑制间隔物其余部分的起始。实际上，起始频率增加了。因此，主复制子并不驻留在原点本身。然而，激活的DHFR启动子是最大起源活性所必需的，如果没有它，复制就会在现在失活的基因中开始。此外，该基因的3'加工信号通过读透转录防止了起源的失活。我们提出，哺乳动物基因组每间隔< 1kb就散布着退化复制子，其活动受局部转录和染色质结构调节。具体目的是：1)确定被认为代表真正复制因子的人层粘胶蛋白B2和-珠蛋白起源是否本质上比DHFR起始区最活跃的位点更活跃；ori-¿将两侧有不匹配的LoxP位点，并与两个人类起源交换，或者与不能有效地启动的片段交换；然后对每一种活动进行量化；层粘连蛋白B2和¿-珠蛋白也将在人类细胞中与驻留在起始区片段进行比较；2)确定ori-¿和ori-？比间隔器中的其他位点更活跃，因为它们的位置相对于局部转录单位；通过ROKO方法，在间隔器中放置不同的ori-¿，并评估对原点效率的影响；3)确定DHFR和较弱的2BE2121启动子是否在起源激活中发挥必要但冗余的作用；这两个启动子都将被删除，并将评估对原产地活性的影响；此外，一个强烈的转录停止信号将被放置在DHFR启动子的下游，以确定转录叉运动（与启动子本身相反）是否需要原点刺激；4)确定了当基因间间隔段被截断时起始效率提高的原因，并制备“超级”起源；将缩短间隔的长度以确定最佳大小，并将一个强转录终止子置于DHFR和2BE2121基因的下游；5)定义通过删除5′和3′调控元件失活或通过上述策略之一激活ori-¿时发生的表观遗传变化；ChIP技术将用于确定起始蛋白和修饰组蛋白的分布，作为Aims 1-4中阐述的变体中细胞周期的功能；这些研究将表明限制起源效率的因素，并将测试通过间隔子转录去除前rcs的预测，而启动子缺失导致无法加载它们。

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.