Replication and Amplification of Tetrahymena rDNA

四膜虫 rDNA 的复制和扩增

• 批准号: 6384113
• 负责人: Geoffrey M. KAPLER
• 金额: $ 25.46万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384113
• 关键词: DNA replication; DNA replication origin; Tetrahymena; cell cycle; eukaryote; gel electrophoresis; gene mutation; genetic mapping; genetic regulatory element; immunoprecipitation; laboratory rabbit; molecular cloning; natural gene amplification; nucleic acid sequence; polymerase chain reaction; replicon; ribosomal DNA; transcription factor

DESCRIPTION (APPLICANT?S ABSTRACT): The organization of eukaryotic replicons is both complex and poorly understood. Whereas regulatory mechanisms assure that chromosomes are duplicated only once per cell division, deviations from strict cell cycle control occur, often leading to the amplification of oncogenes in humans. Organisms that undergo developmentally-programmed gene amplification provide unique opportunities for understanding how replication origins can selectively escape cell cycle control. The Tetrahymena thermophila rDNA minichromosome is an excellent model for dissecting the role of cis-acting determinants, trans-acting factors and chromatin structure in DNA replication control. Tetrahymena rDNA is amplified 5000-fold within a single S phase during development, but is subsequently replicated once per cell cycle. Research in the prior granting period uncovered novel cisacting regulatory determinants for DNA replication, including one that may be dedicated solely to gene amplification. Specific Aim 1 will further dissect the rDNA replicon, and determine the underlying role for newly discovered cis-acting elements in gene amplification and cell cycle-controlled DNA replication. Our biochemical studies have led to the discovery a complex regulatory pathway, in which cell cycle-regulated proteins (TIF 1-4) compete in vivo for binding to an essential rDNA replication determinant. In Aims 2 and 3, we propose reverse genetic approaches to elucidate the role of these proteins and Tetrahymena ORC in rDNA replication control. In Aim 4, we will investigate a developmentally-programmed switch that occurs during gene amplification to generate a strong replication fork barrier. These studies will include examining the role of cis-acting determinants in fork barrier formation, identifying proteins that mediate fork arrest, and exploring the role of chromatin remodeling in amplification of the rDNA minichromosome. The complexity of replicons in higher eukaryotes demonstrates the need to develop non-yeast model systems. Facile forward and reverse genetic approaches in Tetrahymena will be exploited to identify and explore the role of cis-acting determinants and trans-acting factors in the regulation of eukaryotic DNA replication, including underlying mechanisms for gene amplification.

描述（申请人？摘要）：真核生物复制子的结构是 既复杂又不为人知。尽管监管机制确保， 染色体在每次细胞分裂中只复制一次，这与严格的 细胞周期控制发生，通常导致癌基因的扩增， 人类经历发育程序性基因扩增的生物体 提供了独特的机会，了解复制起点如何 选择性地逃避细胞周期控制。嗜热四膜虫rDNA 微型染色体是剖析顺式作用的绝佳模型 DNA复制中的决定簇、反式作用因子和染色质结构 控制四膜虫rDNA在一个S期内扩增5000倍， 发育，但随后每个细胞周期复制一次。研究 前一个授予期发现了新的顺式调节决定因素， DNA复制，包括可能仅用于基因的复制 放大具体目标1将进一步剖析rDNA复制子， 确定新发现的基因顺式作用元件的潜在作用 扩增和细胞周期控制的DNA复制。我们的生化 研究发现了一种复杂的调节途径，在这种途径中， 细胞周期调节蛋白（TIF 1-4）在体内竞争结合必需的 rDNA复制决定簇。在目标2和3中，我们提出了反向遗传 阐明这些蛋白质和四膜虫ORC在rDNA中的作用的方法 复制控制在目标4中，我们将研究一种发展性编程 在基因扩增过程中发生的开关，以产生强复制 分叉屏障这些研究将包括检查顺式作用的作用， 叉屏障形成的决定因素，确定介导叉的蛋白质 抑制，并探讨染色质重塑在扩增中的作用。 rDNA微型染色体。高等真核生物复制子的复杂性 证明了需要开发非酵母模型系统。向前冲， 四膜虫的反向遗传方法将被用来识别和 探讨顺式作用决定簇和反式作用因子在 真核DNA复制的调控，包括 基因扩增