GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

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）染色体断裂和新的端粒 形成，2）内部DNA缺失或DNA剪接，和3）核糖体RNA 基因扩增 这些过程受到高度调控， 在特定的染色体位置，在一个确定的时期， 发展 它们在核分化中起重要作用， 对于理解这种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 分析了参与这些过程的DNA，并开发了第一个DNA 纤毛虫的转化方法。 我们已经开始引入工程化的 利用这种方法，我们将DNA重新注入发育中的细胞核， 发现了指定染色体断裂位点的15 bp序列， 位点特异性DNA缺失所必需的多嘌呤序列，以及42 bp的 反向重复序列对回文rDNA的形成很重要， 放大 我们将继续这种做法，并确定确切的 顺式作用序列需要这些和其他DNA改变过程。 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可能需要哪些其他序列 删除和指定其网站;反向重复如何工作，以促进 回文形成;以及哪些序列负责调节 核糖体基因复制和扩增。 这些结果应该有助于 我们决定其潜在机制的具体方面。 我们将 进一步分离蛋白质或其它大分子， 从同步发育的细胞，并建立在体外反应 系统来确定所涉及的步骤和组件。 通过组合这些 我们希望通过两种方法来了解这些疾病的分子机制， 有趣的遗传过程 它们关系到我们对 许多人类疾病包括癌症。

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.