Analysis of Human Centromeres using Novel Artificial Chromosome Vectors

使用新型人工染色体载体分析人类着丝粒

• 批准号: 7391601
• 负责人: Huntington F Willard
• 金额: $ 28.78万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391601
• 关键词: Artificial Chromosomes; Base Sequence; Biological Assay; Cell Division Process; Cells; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Chromosomes, Artificial, Human; Classification; Code; Cultured Cells; DNA Sequence; Ensure; Epigenetic Process; Event; Genetic; Genome; Genomics; Human; Human Chromosomes; Human Genome; Knowledge; Location; Maps; Meiosis; Mitosis; Nature; Organism; Patients; Plants; Primates; Proteins; Regulation; Role; Signal Transduction; Specific qualifier value; Structure; Technology; Testing; base; design; functional genomics; improved; insight; mammalian genome; mouse genome; novel; research study; segregation; size; vector

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.

描述（由申请人提供）：中心粒是真核染色体的关键组成部分，在确保有丝分裂和减数分裂的适当分离方面具有关键作用。尽管在大多数生物体中精确确定和维持了丝粒的位置，但包括人类基因组在内的许多真核基因组中的丝粒规范的基础是晦涩的，并且可能涉及基于表观遗传和基于序列的事件。中心粒代表了进化悖论：尽管它们在染色体分离中具有重要功能，并且在细胞分裂过程中涉及的许多蛋白质的高度保守性质，但潜在的基因组序列在物种之间和物种之间都高度可变。在人类基因组中，中心粒的特征是大量重复的DNA序列（卫星）的阵列。虽然遗传，基因组和功能研究表明，卫星序列参与人类细胞中的丝粒功能，但序列是高度异质性的，并且与非青春期物种的卫星DMA具有共同的特征。因此，尽管表观遗传调节在指定丝粒染色质方面具有明显的作用，但我们对基因组序列在Centromere规范中的作用的不良理解仍然是当前知识的显着差距。此处描述的实验具有两个具体的目的：（i）改善和验证新型人工染色体技术，以生成在结构上定义的，单位大小的人造染色体，以维持输入矢量序列的大小和结构并可以从人类细胞中恢复，以进行详细分析； （ii）使用人类人造染色体来系统地评估基因组序列及其在培养细胞中的丝粒规范中的作用，通过改变人类A卫星重复单元中的特定序列，通过设计和测试新颖的多中材A卫星阵列阵列，并在全部或一部分中替代了其他型Mammamal seclelien seclelien seclelien sectere and secter sectere sepere sectere and secter sectere satellite sectere and secter secters sectere。这些实验将使我们能够探索尽管缺乏刚性序列保护，但指定了Centromer的身份和功能的基因组代码的性质，并提供了对有助于人类染色体中共晶功能的基因组和表观遗传机制的见解。