Site-Specific DNA Deletion in Tetrahymena thermophila

[600715]考克斯纤毛虫有两个核，一个营养大核和一个种系微核。大核是由微核通过生殖系染色体的一系列程序化断裂事件、某些序列的删除和剩余DNA的扩增而产生的。有关这一过程的机制以及缺失反应的生物学功能的信息有限。我们将开发一种用于这两种发育程序DNA缺失反应的体外系统，即纤毛虫四膜虫（Tetrahymena thermomila）的M区和R区缺失。该实验室之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，然后在体外进行检测以检测预计重组过程的各个步骤。为了便于纯化检测到的任何相关酶，将开发同步交配细胞大规模培养的方法。最终目标是通过研究纯化酶和底物重建的体外系统，对缺失途径进行完整的分子描述。程序性DNA重排不仅是哺乳动物基因组机制（如免疫球蛋白基因）的正常组成部分，而且也是异常基因组现象（如肿瘤发生）的一部分。了解简单单细胞生物（如四膜虫）基因组重组的生物化学过程，可以深入了解人类等更高级生物利用的机制。＊＊＊

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. ***