Genomic Mapping of Replication Origins in Higher Eukaryotes by Okazaki Analysis

通过冈崎分析进行高等真核生物复制起点的基因组作图

• 批准号: 7140232
• 负责人: ZHIFENG SHAO
• 金额: $ 14.79万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140232
• 关键词: DNA replication; DNA replication origin; genetic mapping; molecular biology information system; species difference

DESCRIPTION (provided by applicant): DNA replication is one of the most fundamental biological processes. Starting from multiple sites (replication origins) encoded mostly in non-coding regions of the genome, the completion of duplication of the entire genome is highly regulated and essential for the survival of any species. Despite the very basic nature of this process, less than a few dozen of replication origins in metazoan cells have been characterized to date. Our knowledge on the number of origins, the sites of replication termination and the arrangement of replicons in the genome remains very limited. Here, we propose to develop a versatile, genomic scale mapping method based on strand analysis of Okazaki fragments using short, sequence specific tags generated by lype Us en/ymes. Using the budding yeast as a tractable model system, the detailed procedures of this approach will be validated and optimized. With this method, not only are the replication origins mapped to a high resolution in parallel, but also the fork termination sites that are required to define each individual replicons in the genome. To date, it has not been possible to map the sites of fork termination directly on a large scale. To apply this approach to the much larger human genome containing considerable amount of repeat sequences, the later half of this application is focused on the development of effective protocols to isolate Okazaki fragments on a select region of the genome with the majority of repeat sequences removed. This reduced complexity is required to achieve unique matching of tags of limited length to target sequences. For this purpose, a portion of the sequences selected by the ENCODE project will be used. When these objectives are accomplished, the feasibility of this approach will have been fully demonstrated, and large scale mapping of the human genome, as well as other metazoan genomes, can be initiated with confidence. Among many possible utilities of such comprehensive datasets is the comparative examination of various metazoan genomes that will most certainly reveal the organization and conservation of replicons in the chromosomal context, further elucidating the evolution of one of the most important functional signals embedded in noncoding regions of the genome. It is probable that when the number of known origins increases, consensus sequence motifs or signatures may also be discovered, which can play critical roles in defining and regulating the function of replication origins.

描述（由申请人提供）：DNA复制是最基本的生物学过程之一。从多个位点（复制起源）开始，主要是在基因组的非编码区域中编码的，整个基因组的重复的完成是高度调节的，对于任何物种的存活至关重要。尽管此过程具有非常基本的性质，但迄今为止，在后生动物细胞中不到几十个复制起源。我们对起源数量，复制终止的位点以及基因组中复制子的排列的了解仍然非常有限。在这里，我们建议使用lype us en/ymes生成的简短序列特定标签对冈扎基片段的链分析开发一种多功能的基因组比例映射方法。将芽酵母作为可处理的模型系统，将验证和优化此方法的详细过程。使用这种方法，不仅复制起源在并联中映射到高分辨率，而且是定义基因组中每个单个复制子所需的叉端位点。迄今为止，不可能直接大规模地绘制叉子终止的位置。为了将这种方法应用于包含相当数量的重复序列的更大的人类基因组，该应用的后半部分集中于开发有效方案，以分离基因组的精选区域的Okazaki片段，并消除了大多数重复序列。需要这种降低的复杂性来实现有限长度与目标序列的唯一匹配。为此，将使用编码项目选择的一部分序列。完成这些目标后，该方法的可行性将得到充分证明，人类基因组以及其他后生基因组的大规模映射可以自信地启动。在这种综合数据集的许多可能的实用程序中，有对各种后生基因组的比较检查，这些基因组肯定会揭示在染色体环境中复制子的组织和保护，进一步阐明了嵌入在基因组非编码区域中最重要的功能信号之一。当已知起源的数量增加时，也可能会发现共识序列图案或签名，这可能会在定义和调节复制起源的功能方面起关键作用。