Characterization of TRF-like genes in Arabidopsis

该项目的总体目标是阐明模式植物拟南芥的端粒结构。端粒是重要而复杂的核蛋白结构，在线性真核染色体的物理末端形成保护帽。Shippen实验室先前的研究表明，拟南芥具有显著的可塑性发育和对基因组扰动的耐受性，这使得研究端粒相关基因突变的后果在其他物种中是致命的。本研究的具体目标是鉴定和表征结合双链端粒DNA束的蛋白质。在人类中，这类蛋白质已被证明可以调节端粒长度，防止端到端染色体融合。在BLAST搜索中，Shippen实验室在拟南芥中发现了11个与人类双链端粒蛋白TRF1和TRF2相似的基因。拟南芥基因被命名为trf样基因（TRFL）。对于目标1，将检查TRFL蛋白的DNA结合特性以及它们与其他生物体中已知与端粒相关的蛋白质的相互作用。对于目标2，将确定TRFL蛋白的亚细胞定位，以确定这些成分是否与体内端粒DNA共定位。目的3将通过研究TRFL基因在突变体中失活的后果来研究其在端粒长度维持和基因组稳定性中的功能。对于目标4，将进行TRFL-4的调节和功能。TRFL-4是一种新的基因，其表达受替代mRNA剪接控制，并导致发育调节与端粒DNA的相互作用。由于端粒及其相关蛋白在整个进化过程中都是保守的，这些研究可能为广泛的真核生物提供有助于基因组稳定的基本机制。该项目还将使本科生和研究生有机会在基因组研究的前沿工作的同时，获得植物分子生物学、细胞遗传学和生物化学方面的宝贵专业知识。

The overall goal of this project is to elucidate the architecture of telomeres in the model plant, Arabidopsis thaliana. Telomeres are essential and complex nucleoprotein structures that form protective caps on the physical ends of linear eukaryotic chromosomes. Previous studies by the Shippen lab indicate that Arabidopsis has remarkably plastic development and tolerance to genome perturbations allowing investigations of the consequences of mutations in telomere-related genes that are lethal in other species. The specific objectives of this research focus on identification and characterization of proteins that bind double-strand telomeric DNA tracts. In humans, such proteins have been shown to regulate telomere length and protect against end-to-end chromosome fusions. In a BLAST search, the Shippen lab identified eleven genes in Arabidopsis with features similar to the human double-strand telomere proteins TRF1 and TRF2. The Arabidopsis genes were designated TRF-like (TRFL). For Objective 1, the DNA binding properties of TRFL proteins will be examined as well as their interactions with proteins known to associate with telomeres in other organisms. For Objective 2, the subcellular localization of TRFL proteins will be determined to ascertain whether these components co-localize with telomeric DNA in vivo. Objective 3 will examine the function of TRFL genes in telomere length maintenance and genome stability by studying the consequences of their inactivation in mutants. For Objective 4, regulation and function of TRFL-4 will be undertaken. TRFL-4 is a novel gene whose expression is controlled by alternative mRNA splicing and leads to developmentally regulated interactions with telomeric DNA. Because telomeres and their associated proteins are conserved across evolution, these studies may provide insight into fundamental mechanisms that contribute to genome stability for a broad range of eukaryotic organisms. The project will also give students, both undergraduate and graduate, the opportunity to gain valuable expertise in plant molecular biology, cytogenetics, and biochemistry, while working at the cutting edge of genome research.