GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

四膜虫的基因扩增和消除

• 批准号: 2174647
• 负责人: MENG-CHAO H YAO
• 金额: $ 31.19万
• 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 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174647
• 关键词: DNA binding protein; DNA replication; Tetrahymena; cell transformation; chromosome aberrations; developmental genetics; gel mobility shift assay; gene deletion mutation; gene rearrangement; genetic regulatory element; genome; laboratory rabbit; microorganism genetics; mutant; natural gene amplification; nucleic acid sequence; ribosomal DNA; ribosomal RNA; telomere; transcription factor; western blottings

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.

程序性 DNA 重排是重要的发育过程 发生在多种生物体中，并且与 对包括癌症在内的许多人类疾病的了解。该提案将 检查模型中三个此类过程的分子机制 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。这些过程都受到精确调控， 并且可以被诱导在大量人群中同步发生。过去的 研究揭示了有趣的核苷酸序列特征 负责这些流程的监管。在这个提案中 将采取多种方法分析其顺式作用序列 和反式作用因子，部分依赖于 DNA 转化方法 之前在该实验室开发的。染色体断裂是一个事件 切割 DNA 并向自由端添加新的端粒序列。它 发生在 50-200 个包含 15 bp 序列的特定基因组位点 已知是该过程的信号。在本提案中，尝试将 建立体外反应体系进行细节分析 反应步骤，并识别蛋白质或其他大分子 涉及。此外，还将创建一种特殊的转基因菌株 用于筛选在此过程中有缺陷的突变体。分析这些 突变体将帮助我们了解其调控和生物学作用。脱氧核糖核酸 缺失是一个复杂的过程，发生在数千个基因组中 地点。删除的 DNA 的大小和序列各不相同，并且 总共约占基因组的 15%。对此类删除的研究 element have revealed two types of essential cis-acting sequences: a pair 侧翼调节序列和一组内部激活 序列。现在建议进行研究来定义这些控制 序列，找出它们如何指定删除位点，并学习 如果相同的规则适用于其他删除元素。体外反应 还将建立系统来识别反应中间体并分离 反式作用因子。此外，还将努力描绘 在此发育阶段特异性表达的蛋白质。 最近纯化了一种这样的蛋白质，并显示出有趣的结果 暗示其参与此过程的属性。最后， 核糖体RNA基因的扩增将通过解剖进行分析 其顺式作用序列。该基因被从染色体上切除 在开发过程中被高度放大，并作为免费传播 营养生长过程中的分子。一个特殊的变换向量有 已开发并将用于定义所需的最小序列 用于在生长过程中复制该分子，并揭示其他 发育过程中参与其扩增的序列。这些研究 应该对这些有趣的过程提供新的见解并揭示它们的 监管机制。他们还可以帮助我们了解他们的可能性 与其他细胞过程的关系，例如染色体 凝结、有丝分裂或减数分裂，这些细胞核在 DNA 重排。