GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

四膜虫的基因扩增和消除

• 批准号: 2021809
• 负责人: MENG-CHAO H YAO
• 金额: $ 32.99万
• 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/2021809
• 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.

程序化DNA重排是重要的发育过程， 发生在各种各样的生物体中，并且与 了解包括癌症在内的许多人类疾病。这项建议会 研究模型中三个过程的分子机制 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控制， 并且可以被诱导在大种群中同步发生。过去 研究揭示了有趣的核苷酸序列特征， 负责管理这些过程。在本提案中， 采用多倾向性方法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。染色体断裂 切割DNA并在游离端添加新的端粒序列。它 发生在50-200个特定的基因组位点，包含15 bp序列 这就是这个过程的信号。在这项提议中， 建立体外反应体系，详细分析 反应步骤，并确定蛋白质或其他大分子 涉案此外，还将创建一种特殊的转基因菌株， 用于筛选在这一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。DNA 缺失是一个复杂的过程，发生在数千个基因组中， 地点缺失的DNA在大小和序列上是不同的， 占了基因组的15%研究一个这样的删除 元件揭示了两种类型的基本顺式作用序列：一对 侧翼调控序列和一组内部激活 序列的现在提出的研究，以确定这些控制 序列，找出它们如何指定缺失位点，并学习 如果相同的规则适用于其他删除元素。体外反应 还将建立系统来鉴定反应中间体和分离 反式作用因子此外，还将努力描述 在这个发育阶段特异性表达的蛋白质。 其中一种蛋白质已被纯化，并显示出有趣的 这意味着它参与了这个过程。最后， 核糖体RNA基因的扩增将通过解剖 顺式作用序列该基因从染色体上切除， 在发育过程中高度扩增， 植物生长过程中的分子。一个特殊的变换向量有 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，并揭示其他 在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。它们还可以帮助我们了解 与其他细胞过程的关系，如染色体 浓缩，有丝分裂或减数分裂，这在这些核中从未发生过， DNA重排。