GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

我们建议研究三个相关分子的分子机制。 纤毛原生动物改变基因组结构的过程 四膜虫。这些事件是染色体断裂和新的端粒 形成，2)内部DNA缺失或DNA剪接，以及3)核糖体RNA 基因扩增。这些过程是高度受监管的，发生在 在规定的时期内在特定的染色体位置上可重复 发展。它们在核分化中发挥着重要作用，并且 对于理解这种和其他真核生物的基因组稳定性至关重要。 在过去的授权期内，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。我们已经开始引进工程化的 使用这种方法将DNA重新注入发育中的细胞核，通过这种方法，我们已经 发现了一个15个碱基的序列，它指定了染色体的断裂位置， 位点特异性DNA缺失所需的多嘌呤序列和42bp 反向重复序列在回文rDNA形成中的重要作用 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个BP的不同部分是否 序列负责DNA切割和DNA切割的位点特异性 端粒的形成；促进DNA的其他序列可能需要什么 删除和指定其站点；反向重复如何促进 回文形成；以及哪些序列负责调节 核糖体基因复制和扩增。这些结果应该会有所帮助 美国决定了它们潜在机制的具体方面。我们会 进一步分离执行这些任务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 系统以确定所涉及的步骤和组件。通过将这些组合起来 我们希望通过两种方法来了解这些疾病的分子机制 耐人寻味的遗传过程。它们与我们对 包括癌症在内的许多人类疾病。

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.