GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

四膜虫的基因扩增和消除

• 批准号: 2174646
• 负责人: MENG-CHAO H YAO
• 金额: $ 29.76万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174646
• 关键词: DNA replication; Tetrahymena; cell growth regulation; cell transformation; cytogenetics; developmental genetics; eukaryote; gene deletion mutation; gene rearrangement; genetic manipulation; genetic regulatory element; genome; microinjections; microorganism genetics; natural gene amplification; nucleic acid hybridization; nucleic acid sequence; telomere

We propose to study the molecular mechanisms of three related molecular processes which alter genome structure in the ciliated protozoan Tetrahymena. These events are 1) chromosome breakage and new telomere formation, 2) Internal DNA deletion or DNA splicing, and 3) ribosomal RNA gene amplification. These processes are highly regulated, occurring reproducibly at specific chromosomal locations during a define period of development. They play important roles in nuclear differentiation, and are crucial for understanding genome stability in this and other eukaryotes. In the past granting period we have completed detailed nucleotide sequence analysis of DNAs involved in these processes, and developed the first DNA transformation method for a ciliate. We have begun to introduce engineered DNAs back into developing nuclei using this method, through which we have found a 15 bp sequence which specifies chromosome breakage sites, polypurine sequences necessary for site specific DNA deletion, and a 42 bp inverted repeats important for the formation of palindromic rDNA during amplification. We will continue this approach, and determine the exact cis-acting sequences required for these and other DNA-altering processes. Specifically, we will determine whether different parts of the 15 bp sequence are responsible for the site specificities of DNA cutting and telomere formation; what other sequences may be required for promoting DNA deletion and specifying its sites; how the inverted repeats work to promote palindrome formation; and what sequences are responsible for regulating ribosomal gene replication and amplification. These results should help us determine specific aspects of their underlying mechanisms. We will further isolate proteins or other macro-molecules which carry out these processes from synchronous developing cells, and set up in vitro reaction systems to determine the steps and components involved. By combining these two approaches we hope to understand the molecular mechanisms of these intriguing genetic processes. They are relevant to our understanding of many human diseases including cancer.

我们建议研究三个相关分子的分子机制 改变纤毛原生动物基因组结构的过程 四膜虫。 这些事件是 1) 染色体断裂和新端粒 形成，2) 内部 DNA 缺失或 DNA 剪接，以及 3) 核糖体 RNA 基因扩增。 这些过程受到严格监管，发生 在特定时期内可重复地在特定染色体位置 发展。 它们在核分化中发挥着重要作用， 对于理解这种真核生物和其他真核生物的基因组稳定性至关重要。 在过去的授权期间我们已经完成了详细的核苷酸序列 分析参与这些过程的 DNA，并开发出第一个 DNA 纤毛虫的转化方法。 我们已经开始引入工程化 使用这种方法使 DNA 回到发育中的细胞核，通过这种方法，我们 发现了一个 15 bp 的序列，它指定了染色体断裂位点， 位点特异性 DNA 删除所需的多聚嘌呤序列，以及 42 bp 反向重复序列对于回文 rDNA 的形成很重要 放大。 我们将继续这种方法，并确定确切的 这些和其他 DNA 改变过程所需的顺式作用序列。 具体来说，我们将确定 15 bp 的不同部分是否 序列负责 DNA 切割的位点特异性 端粒形成；促进 DNA 可能还需要哪些其他序列 删除并指定其网站；倒转重复序列如何促进 回文结构；以及哪些序列负责调节 核糖体基因复制和扩增。 这些结果应该有帮助 我们确定其基本机制的具体方面。 我们将 进一步分离执行这些功能的蛋白质或其他大分子 来自同步发育细胞的过程，并建立体外反应 系统来确定所涉及的步骤和组件。 通过结合这些 我们希望通过两种方法来了解这些的分子机制 有趣的遗传过程。 它们与我们的理解相关 许多人类疾病，包括癌症。