Nuclear Structure and Metazoan Origins of Replication

9505907吉尔伯特本实验室成功开发了第一个无细胞系统，其中DNA复制优先在生理上利用的复制起始处启动。将使用细胞和分子生物学工具的组合对这些初步观察进行充分调查，以确定指定复制起点的核结构组件。具体目标是：1.确定在核组装时是否指定了复制起点，2.确定G1核中预先指定的起点是否被非洲爪哇早期卵裂有丝分裂周期破坏，3.确定哺乳动物核膜是否是非洲爪哇卵提取液中定点启动复制所必需的。DNA复制是每个活着的生物体生命周期的核心，虽然人们对简单生物体的复制过程已经知道很多，但高等真核生物中的调控机制在很大程度上是未知的。DNA复制始于真核细胞中确定的染色体位置，但从后生生物衍生的系统中的裸露DNA没有任何这样的要求。这一建议描述了一种新的方法，该方法基于这样的假设，即后生动物染色体中的DNA复制由核结构的各个方面调节，而当从克隆的DNA模板开始时，核结构不容易重建。非洲爪哇卵的无细胞提取物提供了一个很好的模型系统，用来检验细胞核的结构和功能组织在调控DNA复制中至关重要的假设。*？？

9505907 Gilbert Successful development of the first cell-free system in which DNA replication is preferentially initiated at a physiologically utilized origin of replication has been achieved by this laboratory. These initial observations will be fully investigated by using a combination of cell and molecular biological tools to identify the components of nuclear structure that designate an origin of replication. Specific aims are: 1. To determine if origins of replication are designate at the time of nuclear assembly, 2. To determine if pre-designated origins in Gl nuclei are disrupted by a Xenopus early cleavage mitotic cycle, 3. To determine if the mammalian nuclear membrane is necessary for site-specific initiation of replication in Xenopus egg extracts. %%% DNA replication is central to the life cycle of every living organism and, although much is known about the process in simple organisms, the regulatory mechanisms in higher eukaryotes are largely unknown. DNA replication initiates within defined chromosomal loci in eukaryotic cells but naked DNA in systems derived from metazoan organisms do not have any such requirements. This proposal describes a novel approach based upon the hypothesis that DNA replication in metazoan chromosomes is regulated by aspects of nuclear structure that cannot be easily reconstructed when starting with cloned DNA templates. Cell-free extracts from Xenopus eggs provide an excellent model system in which to test the hypothesis that the structural and functional organization of the nucleus is crucial in regulating DNA replication. *** ??