Analysis of Human Centromeres using Novel Artificial Chromosome Vectors

描述（由申请人提供）：着丝粒是真核细胞染色体的关键组成部分，在确保有丝分裂和减数分裂中的正确分离中起关键作用。虽然在大多数生物体中着丝粒的位置被精确地确定和维持，但在许多真核基因组（包括人类基因组）中着丝粒特化的基础是模糊的，并且可能涉及表观遗传和基于序列的事件。着丝粒代表了一个进化悖论：尽管它们在染色体分离中具有重要功能，并且参与细胞分裂过程的许多蛋白质具有高度保守性，但潜在的基因组序列在物种内和物种之间都是高度可变的。在人类基因组中，着丝粒的特征是由串联重复的DNA序列组成的大阵列，即卫星。虽然遗传学、基因组学和功能研究已经证明卫星序列参与人类细胞中的着丝粒功能，但是该序列是高度异质的，并且与非灵长类物种的卫星DMA具有很少的共同特征。因此，尽管表观遗传调控在指定着丝粒染色质的明确作用，我们对基因组序列在着丝粒规范中的作用的理解不足，仍然是目前知识的一个显着差距。（i）改进和验证新的人类人工染色体技术，以产生结构上可定义的单位大小的人类人工染色体，其保持输入载体序列的大小和结构，并且可以从人类细胞中回收用于详细分析;和（ii）使用人人工染色体系统地评价基因组序列及其组织在培养细胞中的着丝粒特化中的作用，通过改变人α卫星重复单元内的特定序列，通过设计和测试新的多聚体α卫星阵列构型，以及通过全部或部分取代来自其它灵长类动物和小鼠基因组的其它哺乳动物着丝粒卫星序列。这些实验将使我们能够探索基因组密码的性质，尽管缺乏严格的序列保守性，指定着丝粒的身份和功能，以及提供见解的基因组和表观遗传机制，有助于人类染色体中的着丝粒功能。

DESCRIPTION (provided by applicant): Centromeres are critical components of eukaryotic chromosomes, with a key role in ensuring proper segregation in mitosis and meiosis. While the location of the centromere is precisely determined and maintained in most organisms, the basis for centromere specification in many eukaryotic genomes, including the human genome, is obscure and likely involves both epigenetic and sequence-based events. Centromeres represent an evolutionary paradox: despite their essential function in chromosome segregation and the highly conserved nature of many proteins involved in the process of cell division, the underlying genomic sequences are highly variable, both within and between species. In the human genome, centromeres are characterized by large arrays of a tandemly repeated DNA sequence, a satellite. While genetic, genomic and functional studies have demonstrated that a satellite sequences are involved in centromere function in human cells, the sequences are highly heterogeneous and share few features in common with satellite DMAs of non-primate species. Thus, notwithstanding a clear role for epigenetic regulation in specifying centromeric chromatin, our poor understanding of the role of genomic sequences in centromere specification remains a significant gap in current knowledge. The experiments described here have two specific aims: (i) to improve and validate novel human artificial chromosome technology to generate structurally definable, unit-sized human artificial chromosomes that maintain the size and structure of the input vector sequences and can be recovered from human cells for detailed analysis; and (ii) to use human artificial chromosomes to systematically evaluate the role of genomic sequences and their organization in centromere specification in cultured cells, by altering specific sequences within the human a satellite repeat unit, by designing and testing novel multimeric a satellite array configurations, and by substituting in whole or in part other mammalian centromeric satellite sequences from both other primate and the mouse genomes. These experiments will allow us to explore the nature of the genomic code that specifies centromere identity and function despite lack of rigid sequence conservation, as well as provide insights into the genomic and epigenetic mechanisms that contribute to centromere function in human chromosomes.