REPLICATION AND AMPLIFICATION OF TETRAHYMENA RDNA

四膜虫 RDNA 的复制和扩增

• 批准号: 6019114
• 负责人: Geoffrey M. KAPLER
• 金额: $ 10.81万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019114
• 关键词: DNA replication; DNA replication origin; Tetrahymena; cell cycle; gel electrophoresis; gene mutation; genetic mapping; genetic regulatory element; molecular cloning; natural gene amplification; nucleic acid sequence; ribosomal DNA; transcription factor

The initiation of DNA replication is normally under cell cycle control. Mechanisms that prevent reinitiation from a replication origin in a given S phase are not understood. In mammals, errors in replication control can result in spontaneous amplification of segments of the genome. Gene amplification is frequently involved in tumorigenesis and, therefore, of significant medical importance. Because amplification events are spontaneous in mammals, our understanding of amplification mechanisms is limited. In contrast, gene amplification is developmentally-programmed in the protozoan Tetrahymena, providing the opportunity to directly study and dissect the amplification process. The rDNA minichromosome of T. thermophila, encoding the ribosomal RNA genes, has been used extensively as a model for DNA replication and gene amplification. Vegetative replication of the rDNA initiates from a single origin of replication. Cis-acting sequences controlling initiation from this origin have been identified. The vegetative origin is also used to amplify the rDNA. Thus, cell cycle control of this origin is suppressed during gene amplification. During early amplification, the vegetative origin is quiescent. At this time, developmentally-regulated mechanisms promote initiation from other segments of the chromosome. rDNA amplification occurs as part of a developmental program, accompanied by chromosome breakage and DNA rearrangement, similar to mammalian amplification events. Cis-acting mutations affecting excision, rearrangement and amplification of the rDNA have been identified. Their roles in amplification are beginning to be understood. Classical genetic, molecular and physical approaches will be employed to investigate the control of replication and amplification of the rDNA minichromosome. New cis-acting elements controlling the formation and amplification of the rDNA will be identified by DNA sequencing of existing mutants. These mutants will be exploited to identify genes encoding trans- acting factors required for information and amplification of the rDNA. Extragenic suppressors of these recessive-lethal cis-acting mutations will be sought. rDNA replication origins and cis-regulatory determinants will be studied. 2D gel electrophoresis will be used to determine whether early amplification initiates randomly or from specific developmentally- regulated replication origins. Amplification origins will be precisely mapped by 2D methods. Cis-acting elements required for amplification will be localized by deletional analysis of rDNA constructs, using a transient transformation DNA replication assay. These elements should include constitutive DNA replication determinants. Amplification-specific determinants, such as binding sites for factors that suppress cell cycle control, should also be identified. 2D analysis and transformation will be used to further localize the vegetative replication origin and identify the minimal origin of vegetative rDNA replication. Mutants unable to amplify the rDNA will be exploited as DNA transformation recipients for these studies. By understanding the relationship of gene amplification to cell cycle-regulated replication in Tetrahymena, mechanisms that control these processes in higher eukaryotes may become clear.

DNA复制的起始通常受细胞周期控制。 在给定的细胞中阻止从复制起点重新起始的机制 S阶段不被理解。在哺乳动物中，复制控制中的错误可以 导致基因组片段的自发扩增。基因 扩增经常参与肿瘤发生，因此， 重要的医学意义。因为放大事件是 自发的哺乳动物，我们的理解放大机制是 有限公司相比之下，基因扩增是发育编程的， 原生动物四膜虫，提供了直接研究和 剖析放大过程。 T.的rDNA微型染色体。嗜热菌，编码核糖体RNA 基因，已被广泛用作DNA复制和基因表达的模型。 放大rDNA的无性复制起始于一个 复制起点顺式作用序列控制起始自 这个起源已经被确认。植物起源也用于 扩增rDNA。因此，这种起源的细胞周期控制被抑制 在基因扩增过程中。在早期扩增过程中， 起源是静止的。在这个时候，发展调节机制 促进从染色体的其它区段起始。rDNA 扩增作为发育程序的一部分发生，伴随着 染色体断裂和DNA重排，类似于哺乳动物 放大事件影响切除的顺式作用突变， 已经鉴定了rDNA的重排和扩增。他们的 在放大中的作用开始被理解。 将采用经典的遗传、分子和物理方法， 研究rDNA复制和扩增的控制 微型染色体新的顺式作用元件控制的形成和 rDNA的扩增将通过现有的DNA测序来鉴定。 变种人这些突变体将被用来鉴定编码反式- rDNA的信息和扩增所需的作用因子。 这些隐性致死顺式作用突变的基因外抑制因子将 被寻找。rDNA复制起点和顺式调节决定簇将 被研究。2D凝胶电泳将用于确定是否早期 扩增随机启动或从特定的发育- 调节复制起点。放大源将精确地 用2D方法绘制。扩增所需的顺式作用元件将 通过rDNA构建体的缺失分析，使用瞬时 转化DNA复制测定。这些要素应包括 组成型DNA复制决定簇。扩增特异性 决定子，如抑制细胞周期因子的结合位点 控制，也应该被识别。2D分析和转换将是 用于进一步定位营养体复制起点， 植物rDNA复制的最小起点。变种人无法 扩增rDNA将被利用作为DNA转化受体， 这些研究。通过了解基因扩增与 细胞周期调控的四膜虫复制，控制机制 高等真核生物中的这些过程可能变得清楚。