Developmental and genomic studies on the agnatha VLR system

描述(申请人提供)：“基因组学”方法的出现揭示了在后生动物中产生免疫受体谱系的多种系统发育机制。最近在无脊椎动物中描述了一种能够通过躯体重排产生巨大多样性的淋巴细胞抗原受体系统，这两个类群代表了最原始的脊椎动物，但缺乏高等脊椎动物适应性免疫所必需的标志成分，即免疫球蛋白超家族(IgSF)的重排基因。这些可变淋巴细胞受体(VLR)是通过一种全新的分子和遗传机制产生的，在这种机制中，大量的富含亮氨酸重复序列(LRR)盒被用来构建受体分子的多样性区域。在淋巴细胞发育过程中，这些盒通过一种尚未确定的机制从基因组的其他部分输入到一个不完整的生殖系基因中。基于其已知的序列多样性，其推测的三维结构，并暗示其他已知的含有LRR的蛋白质，如Toll样受体，VLRs的胞外结构域被认为能够结合广泛的病原体特异性。这一新系统的存在表明，在脊椎动物中进化适应性免疫系统至少有两种独立的解决方案，进而引发了关于VLR系统的进化和遗传起源以及我们对适应性免疫的概念的许多关键问题。此外，这一发现有助于验证比较免疫学社区所采用的基因组方法，以便对免疫的系统发展做出推断。在R21的应用中，我们关注VLR系统的两个基本方面，即它在早期发育中的作用和它的组件模块的基因组精细结构。我们将定义VLR系统运行的最早开发阶段，涉及哪些蜂窝结构，以及它使用的是与先前定义的相同还是不同的曲目。此外，我们试图确定VLR系统在早期胚胎发育中是否具有替代作用。根据其基因的精细结构，我们试图从基因组上克隆、鉴定和测序海鳗的整个VLR基因座，以便更好地确定该复合体的结构和重排可能发生的方式。

DESCRIPTION (provided by applicant): The advent of "genomics" approaches has unveiled a diversity of phylogenetic mechanisms for generating immune receptor repertoires amongst metazoans. A lymphocyte antigen receptor system capable of generating enormous diversity via somatic rearrangement has recently been described in the agnathans (lamprey and hagfish), taxa representing the most primitive vertebrates but which lack the hallmark components necessary for adaptive immunity in higher vertebrates, i.e., the rearranging genes of the immunoglobulin superfamily (IgSF). These variable lymphocyte receptors (VLRs) are produced through an entirely novel molecular and genetic mechanism in which large banks of leucine rich repeat (LRR) cassettes are used to build the "diversity" region of the receptor molecules. These cassettes are imported into an incomplete germline gene from the other parts of the genome during lymphocyte development through an as-yet undefined mechanism. Based on its known sequence diversity, its inferred three-dimensional structure, and by implication to other known LRR-containing proteins such as the Toll-like receptors, the ectodomains of the VLRs are assumed to be capable of binding a wide range of pathogen specificities. Existence of this novel system suggests that there were at least two independent solutions to evolving an adaptive immune system in vertebrates and, in turn, raises many critical questions with regard to the evolutionary and genetic origins of the VLR system and our conception of adaptive immunity. Moreover, the discovery helps to validate the genomic approaches being embraced by the comparative immunology community in order to make inferences concerning the phylogeny of immunity. In this R21 application, we focus on two fundamental aspects of the VLR system, namely its role in early development and the genomic fine structure of its component modules. We will define the earliest stages in development at which the VLR system is operating, what cellular structures are involved, and whether it uses the same or different repertoire as previously defined. Moreover, we seek to determine whether the VLR system has an alternative role during early embryonic development. In terms of the genomic fine structure of its locus, we seek to genomically clone, characterize and sequence the entire VLR locus of the sea lamprey so as to better define how the complex is structured and how the rearrangements may be occurring.