Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

基因组动力学的调控在进化上是保守的。维持基因组完整性的一个关键因素是无处不在的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，但仍不清楚Rad51p如何介导其作用。本项目主要研究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，基因操作可以显示Rad51p的可分离功能，包括在其他模式生物中未见的作用。纤毛虫拥有一个不寻常的基因组组织，有效地将体细胞和种系遗传功能划分为两个不同的细胞核。微核（MIC）是种系细胞核，在营养生长期间进行有丝分裂，转录沉默。巨核（MAC）是体细胞核，无丝分裂，转录活跃。偶联对的mic经历一系列减数分裂和有丝分裂，随后交换单倍体核，融合产生合子核。在结合后期，亲本mac被破坏，新的mac以一种程序化的方式从合子MIC的拷贝中发育出来。四膜虫RAD51的表达在不同的环境条件和发育阶段有所不同。在营养生长期间，Rad51p的高表达使MAC分裂与细胞周期分离，导致无核后代的高比例。这个项目有两个目标。首先，将筛选cDNA表达文库，筛选抑制组成性表达Rad51p的菌株所表现出的严重无核表型的四膜虫基因产物。这种实验方法将有助于确定在大核分裂过程中直接和/或间接与Rad51p相互作用的因素。其次，随机诱变的Rad51p将组成性表达，以识别减轻转化子无核表型的错义突变。该项目利用仅在四膜虫实验系统中可能的遗传操作，应该扩展我们对Rad51p结构和功能的理解，从而有助于在其他真核生物模型系统中分析这一高度重要的蛋白质。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.