Developmental and genomic studies on the agnatha VLR system

描述（由申请人提供）：“基因组学”方法的出现揭示了后生动物中产生免疫受体库的多种系统发育机制。最近在无颌动物（七鳃鳗和盲鳗）中描述了一种能够通过体细胞重排产生巨大多样性的淋巴细胞抗原受体系统，这种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需的标志性成分，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，其中大量富含亮氨酸的重复序列（LRR）盒用于构建受体分子的“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导入到不完整的种系基因中。基于其已知的序列多样性、推断的三维结构以及与其他已知的含有 LRR 的蛋白质（例如 Toll 样受体）的暗示，VLR 的胞外域被认为能够结合广泛的病原体特异性。这种新系统的存在表明，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，反过来，提出了许多关于 VLR 系统的进化和遗传起源以及我们的适应性免疫概念的关键问题。此外，这一发现有助于验证比较免疫学界所采用的基因组方法，以便对免疫系统发育做出推断。在此 R21 应用中，我们重点关注 VLR 系统的两个基本方面，即其在早期发育中的作用及其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、涉及哪些细胞结构以及它是否使用与之前定义的相同或不同的指令。此外，我们试图确定 VLR 系统在早期胚胎发育过程中是否具有替代作用。就其位点的基因组精细结构而言，我们寻求对海七鳃鳗的整个VLR位点进行基因组克隆、表征和测序，以便更好地定义复合物的结构以及重排是如何发生的。

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.