Site-Specific DNA Deletion in Tetrahymena thermophila

嗜热四膜虫中位点特异性 DNA 缺失

• 批准号: 9600715
• 负责人: Michael Cox
• 金额: $ 16万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9600715&HistoricalAwards=false
• 关键词: Site; Specific; DNA; Deletion; Tetrahymena

9600715 Cox Cilliates possess two nuclei, a vegetative macronucleus and a germline micronucleus. The macronucleus is produced from the micronucleus by a programmed series of fragmentation events of the germline chromosomes, deletion of some sequences and amplification of the remaining DNA. There is only limited information about the mechanisms involved in this process as well as the biological function of the deletion reactions. An in vitro system for two of these developmentally programmed DNA deletion reactions will be developed, the M and R region deletions from the ciliate, Tetrahymena thermophila. Previous work from this laboratory has suggested a likely deletion pathway and a protocol is envisioned in which new DNA substrates are first tested in vivo and then assays are carried out to detect individual steps of the projected recombination process in vitro. To facilitate the purification of any relevant enzymes detected, methods to produce large-scale cultures of synchronously mating cells will be developed. The ultimate goal is a complete molecular description of the deletion pathway derived from studies of an in vitro system reconstituted from purified enzymes and substrates. %%% Programmed DNA rearrangements are not only a normal part of mammalian genome mechanisms (i.e. immunoglobulin genes) but also of aberrant genome phenomena (i.e. tumorogensis). An understanding of the biochemistry of genome restructuring in simple one-celled organisms such as Tetrahymena can give insight into the mechanisms utilized by more advanced organisms such as humans. ***

9600715考克斯 纤毛虫有两个核，一个营养大核和一个生殖微核。大核是通过一系列程序化的生殖系染色体片段化事件、某些序列的缺失和剩余DNA的扩增从微核产生的。关于这一过程中涉及的机制以及缺失反应的生物学功能，只有有限的信息。将开发两个这些发育程序化的DNA缺失反应的体外系统，从纤毛虫，嗜热四膜虫的M和R区缺失。该实验室以前的工作已经提出了一种可能的缺失途径，并设想了一种方案，其中首先在体内测试新的DNA底物，然后进行测定以检测体外预测重组过程的各个步骤。为了便于纯化检测到的任何相关酶，将开发生产同步交配细胞的大规模培养物的方法。最终目标是从纯化的酶和底物重建的体外系统的研究中获得缺失途径的完整分子描述。 程序性DNA重排不仅是哺乳动物基因组机制（即免疫球蛋白基因）的正常部分，也是异常基因组现象（即肿瘤发生）的一部分。了解简单的单细胞生物（如四膜虫）基因组重组的生物化学可以深入了解更先进的生物（如人类）所利用的机制。 ***