Replication and Amplification of Tetrahymena rDNA

描述(申请人？S摘要)：真核复制子的组织是 既复杂又鲜为人知。鉴于监管机制确保 染色体在每个细胞分裂中只复制一次，偏离了严格的 发生细胞周期控制，通常导致癌基因在 人类。经历发育程序性基因扩增的生物体 提供独特的机会，让您了解复制来源如何 选择性地逃避细胞周期控制。嗜热四膜虫rDNA 微染色体是剖析顺式作用作用的极佳模型 DNA复制中的决定因素、反式作用因子和染色质结构 控制力。四膜虫rDNA在单个S时相中扩增5000倍 发育，但随后每个细胞周期复制一次。研究领域 之前的授权期发现了新的顺应性监管决定因素 DNA复制，包括可能专用于基因的复制 放大。《特定目标1》将进一步剖析rDNA复制子，以及 确定新发现的顺式作用元件在基因中的潜在作用 扩增和细胞周期控制的DNA复制。我们的生物化学 研究发现了一种复杂的调控途径，在这种途径中，细胞 循环调节蛋白(TIF 1-4)在体内竞争与必需的 RDNA复制决定因素。在目标2和目标3中，我们提出了反向遗传 阐明这些蛋白和ORC四膜虫在rDNA中作用的方法 复制控制。在目标4中，我们将调查一个发育编程的 在基因扩增过程中发生的一种开关，以产生强大的复制 叉形栅栏。这些研究将包括考察顺式作用的作用。 分叉屏障形成中的决定因素，识别介导分叉的蛋白质 并探讨染色质重塑在细胞增殖中的作用。 RDNA微染色体。高等真核生物中复制子的复杂性 论证了开发非酵母模型系统的必要性。方便地向前和 将利用四膜虫的反向遗传方法来识别和 探索顺式作用决定因素和反式作用因子在 真核细胞DNA复制的调控，包括 基因扩增。

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.