Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

基因组动力学的调节在进化上是保守的。维持基因组完整性的一个关键因素是普遍存在的DNA链转移蛋白Rad51p。尽管从酵母到哺乳动物的模型系统对RAD51进行了整整十年的研究，但仍然不清楚Rad51p是如何调节其影响的。这个项目的重点是纤毛原生动物嗜热四膜虫的RAD51。由于四膜虫独特的细胞生物学和核组织，遗传操作可以展示Rad51p的可分离功能，包括在其他模式生物中未见过的作用。纤毛虫拥有一种不同寻常的基因组组织，它有效地将体细胞和生殖系的遗传功能划分在两个不同的核之间。微核(MIC)是生殖核，在营养生长过程中有丝分裂，转录沉默。大核(MAC)是体细胞核，无丝分裂，转录活跃。接合对的MIC经历了一系列减数分裂和有丝分裂，然后是单倍体核的交换，单倍体核融合产生合子核。在接合后期，亲本的MIC被破坏，新的MIC以编程的方式从受精卵MIC的副本中发育出来。四膜虫RAD51的表达在不同的环境条件和发育阶段不同。在营养生长过程中，RAD51p的结构性高表达使MAC分裂与细胞周期脱钩，导致高比例的无核后代。这个项目有两个目标。首先，将筛选四膜虫基因产物的cdna表达文库，这些基因产物可以抑制组成表达Rad51p的菌株表现出的严重无核表型。这一实验方法将有助于识别在大核分裂过程中直接和/或间接与Rad51p相互作用的因子。其次，随机突变的Rad51p将被结构性表达，以努力识别错义突变，以减轻转化子中的无核表型。该项目利用了仅在四膜虫实验系统中可能进行的遗传操作，应该会扩大我们对Rad51p结构和功能的理解，从而有助于在其他真核模型系统中分析这种非常重要的蛋白质。本科生和研究生都将帮助开展这项研究。

The regulation of genome dynamics has been evolutionarily conserved. A key factor involved in maintaining the genome's integrity is the ubiquitous DNA strand transfer protein Rad51p. Despite a full decade of research on RAD51 from model systems ranging from yeast to mammals, it is still unclear how Rad51p mediates its effects. This project focuses on RAD51 from the ciliated protozoan Tetrahymena thermophila. Due to Tetrahymena's unique cell biology and nuclear organization, genetic manipulations can showcase the separable functions of Rad51p, including roles unwitnessed in other model organisms. Ciliates possess an unusual genome organization that effectively divides the somatic and germ-line genetic functions between two distinct nuclei. The micronucleus (MIC) is the germline nucleus, dividing mitotically during vegetative growth, and transcriptionally silent. The macronucleus (MAC) is the somatic nucleus, dividing amitotically, and transcriptionally active. The MICs of a conjugating pair undergo a series of meiotic and mitotic divisions, followed by an exchange of haploid nuclei, which fuse to produce a zygotic nucleus. Late in conjugation the parental MACs are destroyed, and new MACs develop in a programmed manner from copies of the zygotic MIC. Tetrahymena RAD51 expression varies under a number of different environmental conditions and developmental stages. Constitutively high Rad51p expression during vegetative growth decouples MAC division from the cell cycle, leading to a high percentage of amacronucleate progeny. This project has two goals. First, a cDNA expression library will be screened for Tetrahymena gene products that suppress the severe amacronucleate phenotype exhibited by strains that constitutively express Rad51p. This experimental approach will help identify factors that directly and/or indirectly interact with Rad51p during macronuclear division. Second, randomly mutagenized Rad51p will be constitutively expressed in an effort to identify missense mutations that alleviate the amacronucleate phenotype in transformants. This project, taking advantage of genetic manipulations possible only within the Tetrahymena experimental system, should extend our understanding of Rad51p structure and function and thus will contribute to the analysis of this highly important protein in other eukaryotic model systems. Both undergraduate and graduate students will help carry out this research.