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普遍存在的DNA链转移蛋白Rad 51 p。 尽管从酵母到哺乳动物的模型系统对Rad 51进行了整整十年的研究，但仍不清楚Rad 51 p如何介导其作用。 本项目的重点是从纤毛原生动物四膜虫嗜热四膜虫的RAD 51。 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Rad 51 p的可分离功能，包括在其他模式生物中未被见证的角色。 纤毛虫拥有一个不寻常的基因组组织，有效地划分两个不同的细胞核之间的体细胞和生殖系的遗传功能。 微核（MIC）是生殖细胞核，在营养生长期间进行有丝分裂，并且转录沉默。 大核（MAC）是体细胞核，无丝分裂，转录活跃。 接合对的MIC经历一系列减数分裂和有丝分裂，随后是单倍体核的交换，其融合以产生合子核。 在接合后期，亲本MAC被破坏，并且新的MAC以程序化的方式从合子MIC的拷贝发展。 四膜虫RAD 51的表达在许多不同的环境条件和发育阶段下变化。 营养生长过程中Rad 51 p的组成性高表达使MAC分裂脱离细胞周期，导致高比例的无核后代。 这个项目有两个目标。 首先，将筛选cDNA表达文库中的四膜虫基因产物，其抑制组成型表达Rad 51 p的菌株所表现出的严重的无核表型。 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Rad 51 p相互作用的因素。 第二，随机诱变的Rad 51 p将组成型表达，以鉴定减轻转化体中无短核表型的错义突变。 这个项目，利用遗传操作可能只有在四膜虫实验系统，应该扩展我们的理解Rad 51 p的结构和功能，从而将有助于在其他真核生物模型系统中分析这一非常重要的蛋白质。 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.