GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

程序性DNA重排是重要的发育过程， 发生在各种各样的生物体中，并与 了解包括癌症在内的许多人类疾病。这项提议将 在模型中检查三个这样的过程的分子机制 真核生物嗜热四膜虫。它们是：DNA缺失、染色体 断裂和基因扩增。这些过程受到精确的监管， 并且可以诱导在大种群中同步发生。过去时 研究揭示了有趣的核苷酸序列特征，这些特征 负责这些流程的监管。在本提案中， 将采取多种倾向的方法来分析它们的顺式作用序列 和反式作用因子，部分依赖于DNA转化方法 以前在这个实验室里开发的。染色体断裂是一件大事 这会切割DNA，并在游离端添加新的端粒序列。它 发生在50-200个特定的基因组位置，包含15个碱基序列 已知是这一过程的信号。在本提案中，尝试将 必须建立体外反应系统来分析细节 反应步骤，并识别蛋白质或其他大分子 牵涉其中。此外，还将创造一种特殊的转基因菌株，并 用于筛选在这一过程中有缺陷的突变体。对这些问题的分析 突变体将帮助我们了解它的调控和生物学作用。脱氧核糖核酸 缺失是一个复杂的过程，在几千个基因组中发生 地点。缺失的DNA在大小和序列上是不同的，并且 加在一起约占基因组的15%。对其中一例缺失的研究 元素揭示了两种基本的顺式作用序列：一对 侧翼调控序列和一组内部激活 序列。现在提出了一些研究来定义这些控制 序列，找出它们是如何指定缺失位置的，并学习 如果相同的规则适用于其他删除元素。一种体外反应 还将建立系统来识别反应中间体并分离 反式作用因素。此外，还将努力将 在发育的这一阶段专门表达的蛋白质。 最近提纯了一种这样的蛋白质，并显示出有趣的一面 暗示它参与了这一进程的财产。最后， 核糖体rna基因的扩增将通过解剖进行分析。 ITS顺式作用序列。这个基因被从染色体上切除， 在发育过程中高度放大，作为免费传播 分子在营养生长过程中。一种特殊的变换向量具有 已开发，并将用于定义所需的最小序列 在生长过程中复制这种分子，并发现其他 在发育过程中参与ITS扩增的序列。这些研究 应该为这些有趣的过程提供新的见解，并揭示它们的 监管机制。它们还可能帮助我们理解它们可能 与其他细胞过程的关系，如染色体 凝集、有丝分裂或减数分裂，这些在这些细胞核中从未发生过 DNA重排。

Programmed DNA rearrangements are important developmental processes that occur in a wide variety of organisms, and are relevant to the understanding of many human diseases including cancer. This proposal will examine the molecular mechanisms of three such processes in the model eukaryote Tetrahymena thermophila. They are: DNA deletion, chromosome breakage and gene amplification. These processes are precisely regulated, and can be induced to occur synchronously in large populations. Past studies have revealed interesting nucleotide sequence features that are responsible for the regulation of these processes. In this proposal a multi prone approach will be taken to analyze their cis-acting sequences and trans-acting factors, relying partly on a DNA transformation method developed previously in this laboratory. Chromosome breakage is an event that cleaves DNA and adds new telomeric sequences to the free ends. It occurs at 50-200 specific genomic sites that contain a 15 bp sequence known to be the signal for this process. In this proposal attempts will be made to establish an in vitro reaction system to analyze the details of the reaction steps, and to identify proteins or other macromolecules involved. In addition, a special transgenic strain will be created and used to screen for mutants defective in this process. Analysis of these mutants will help us understand its regulation and biological roles. DNA deletion is a complex process that occurs at several thousand genomic locations. The deleted DNAs are diverse in size and sequence, and together comprise about 15% of the genome. Studies of one such deletion element have revealed two types of essential cis-acting sequences: a pair of flanking regulatory sequences and a set of internal activating sequences. Studies are now proposed to define these controlling sequences, to find out how they specify the deletion site, and to learn if the same rule applies to other deletion elements. An in vitro reaction system will also be set up to identify reaction intermediates and isolate trans-acting factors. In addition, efforts will be made to characterize proteins that are specifically expressed at this stage of development. One such protein has been purified recently and show interesting properties implying its involvement in this process. Finally, amplification of the ribosomal RNA gene will be analyzed by dissecting its cis-acting sequences. This gene is excised from the chromosome and become highly amplified during development, which is propagated as free molecules during vegetative growth. A special transformation vector has been developed and will be used to define the minimal sequence required for the replication of this molecule during growth, and uncover other sequences involved in its amplification during development. These studies should give new insights into these intriguing processes and reveal their regulatory mechanisms. They may also help us understand their possible relationships to other cellular processes, such as chromosome condensation, mitosis or meiosis, which never occur in these nuclei after DNA rearrangements.