Replication of Mammalian Chromosomes

描述（由申请人提供）：DHFR起点由>22个无效起始位点的区域组成，所述无效起始位点散布在会聚转录的DHFR和2BE 2121基因之间的55 kb间隔区中。然而，一些网站（ori-<$and ori-？）显然是优选的。我们已经证明，从DHFR起始点原位缺失最活跃的起始位点，或者甚至包括>90%活性位点的中心40-kb核心，不会抑制间隔区其余部分的起始。事实上，启动频率实际上增加了。因此，主复制子并不存在于起源本身。然而，一个活跃的DHFR启动子是最大的起始活性所必需的，在它的缺失下，复制在现在失活的基因中启动。此外，基因的3'加工信号通过通读转录防止起点失活。我们认为哺乳动物基因组中每隔<1 kb就有简并复制子，它们的活动受局部转录和染色质结构的调节。具体目标是：1）确定被认为代表真正复制子的人核纤层蛋白B2和<$-珠蛋白起源是否本质上比DHFR起始区中最活跃的位点更活跃; ori-<$将与错配的LoxP位点侧接并与两个人起源交换，或与不能有效原位起始的片段交换;然后将定量每个的活性;核纤层蛋白B2和<$-珠蛋白起源的活性。- 珠蛋白也将在人类细胞中进行比较，以片段驻留在起始区; 2）以确定是否ori-<$and ori-？3）确定DHFR和较弱的2BE 2121启动子是否在起始激活中起重要但冗余的作用;两个启动子都将被删除，并将评估对起源活性的影响;此外，将在DHFR启动子的下游放置一个强转录终止信号，以确定转录叉移动是否4）确定当基因间间隔区被截短时起始效率增加的原因，并制备“超级”起始;间隔区的长度将被减少以确定最佳大小，并且强转录终止子将被放置在DHFR和2BE 2121基因的下游; 5）以定义当ori-2121时发生的表观遗传变化。通过缺失5'和3'调控元件失活或通过上述策略之一激活; ChIP技术将用于确定起始蛋白和修饰的组蛋白的分布，作为目标1-4中详述的变体中细胞周期的函数;这些研究将指出限制起源效率的因素，并将检验通过间隔区转录去除前RC，而启动子缺失导致不能加载它们的预测。

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.