Analysis of Human Centromeres using Novel Artificial Chromosome Vectors

描述(申请人提供)：着丝粒是真核细胞染色体的重要组成部分，在确保有丝分裂和减数分裂中的适当分离方面起着关键作用。虽然着丝粒的位置在大多数生物体中是精确确定和保持的，但在许多真核生物基因组中，包括人类基因组在内的着丝粒指定的基础是模糊的，可能涉及表观遗传和基于序列的事件。着丝粒代表了一种进化悖论：尽管它们在染色体分离中具有重要功能，而且许多参与细胞分裂过程的蛋白质具有高度保守性，但其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，着丝粒的特征是一大串重复的DNA序列，即卫星。虽然遗传、基因组和功能研究表明，卫星序列参与了人类细胞的着丝粒功能，但这些序列具有高度的异质性，与非灵长类物种的卫星DMA几乎没有共同的特征。因此，尽管表观遗传调控在指定着丝粒染色质中具有明确的作用，但我们对基因组序列在着丝粒指定中的作用的了解仍然是当前知识中的一个重大缺口。这里描述的实验有两个具体目标：(1)改进和验证新型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，这些染色体保持输入载体序列的大小和结构，并可从人类细胞中回收用于详细分析；(2)利用人类人工染色体，通过改变人类基因组中的特定序列，改变人类卫星重复单位内的特定序列，通过设计和测试新型多聚体卫星阵列配置，以及全部或部分替代来自其他灵长类和小鼠基因组的其他哺乳动物着丝粒卫星序列，来系统地评估基因组序列及其组织在培养细胞着丝粒指定中的作用。这些实验将使我们能够探索基因组密码的性质，该密码指定着丝粒的身份和功能，尽管缺乏严格的序列保守，并提供了对人类染色体着丝粒功能的基因组和表观遗传机制的见解。

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.