REPLICATION AND AMPLIFICATION OF TETRAHYMENA RDNA

四膜虫 RDNA 的复制和扩增

• 批准号: 6313751
• 负责人: Geoffrey M. KAPLER
• 金额: $ 10.98万
• 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 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6313751
• 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复制的启动通常受细胞周期的控制。 防止从给定的复制源重新启动的机制 对S阶段的认识还不够深入。在哺乳动物中，复制控制中的错误可能 导致基因组片段的自发扩增。基因 扩增经常参与肿瘤的发生，因此， 具有重要的医学意义。因为放大事件是 在哺乳动物中自发的，我们对扩增机制的理解是 有限的。相反，基因扩增是发育编程的 原生动物四膜虫，为直接研究和 剖析扩增过程。 嗜热毛滴虫的rDNA微染色体，编码核糖体RNA 基因，已经被广泛地用作DNA复制和基因的模型 放大。RDNA的营养复制始于单个 复制的来源。顺式作用序列控制从 这一起源已被确定。植物起源也被用来 扩增rDNA。因此，该起始点的细胞周期控制被抑制 在基因扩增过程中。在早期的放大过程中，植物性 起源是静止的。在这个时候，发育调节机制 促进染色体其他片段的启动。RDNA 扩增是发育计划的一部分，伴随着 染色体断裂和DNA重排，与哺乳动物相似 放大事件。影响切除的顺式作用突变， 已经确定了rDNA的重排和扩增。他们的 人们开始认识到放大的作用。 将使用经典的遗传、分子和物理方法来 研究rDNA复制和扩增的控制 小染色体。新的顺式作用元件控制形成和 RDNA的扩增将通过现有的DNA测序进行鉴定 变种人。这些突变体将被用来识别编码反式-DNA的基因。 RDNA的信息和扩增所需的作用因子。 这些隐性致死顺式突变的外源抑制子将 被追寻。RDNA复制起点和顺式调控决定因素将 被研究。将使用2D凝胶电泳法确定早期 扩增是随机启动的，或者是从特定的发育阶段- 受管制的复制来源。放大的起源将精确地 通过2D方法进行映射。扩增所需的顺式作用元件将 通过对rDNA结构的缺失分析来定位，使用瞬时 转化DNA复制试验。这些要素应包括 构成DNA复制决定因素。特定于放大的 决定因素，如抑制细胞周期的因子的结合位点 控制，也应该被识别。将进行2D分析和转换 用于进一步定位营养复制起始点并识别 营养rDNA复制的最小起源。变种人无法 扩增rDNA将被用作DNA转化的受体 这些研究。通过了解基因扩增与 四膜虫中细胞周期调节的复制，控制机制 高等真核生物的这些过程可能会变得清晰起来。

项目成果

Transient and stable DNA transformation of Tetrahymena thermophila by electroporation.

• DOI: 10.1016/s0091-679x(08)61552-6
• 发表时间: 2000
• 期刊: Methods in cell biology
• 作者: Jacek Gaertig;Geoffrey M. Kapler

TIF1 activates the intra-S-phase checkpoint response in the diploid micronucleus and amitotic polyploid macronucleus of Tetrahymena.

TIF1 激活四膜虫二倍体微核和无丝分裂多倍体大核中的 S 期内检查点反应。

• DOI: 10.1091/mbc.e06-05-0469
• 发表时间: 2006
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Yakisich,JSebastian;Sandoval,PamelaY;Morrison,TaraL;Kapler,GeoffreyM
• 通讯作者: Kapler,GeoffreyM

Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome.

四心hymena hythyphila rDNA复制叉屏障区域的缺失破坏了大核RDNA切除，并在微核基因组中产生脆弱的位点。

• DOI: 10.1093/nar/gkj466
• 发表时间: 2006
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Yakisich JS;Kapler GM
• 通讯作者: Kapler GM

A beta-tubulin mutation selectively uncouples nuclear division and cytokinesis in Tetrahymena thermophila.

β-微管蛋白突变选择性地解开嗜热四膜虫的核分裂和胞质分裂。

• DOI: 10.1128/ec.3.5.1217-1226.2004
• 发表时间: 2004
• 期刊: Eukaryotic cell
• 作者: Smith,JoshuaJ;Yakisich,JSebastian;Kapler,GeoffreyM;Cole,EricS;Romero,DanielP
• 通讯作者: Romero,DanielP