Immunoglobulin gene expression and recombinational events in the nurse shark

淋巴细胞表达大量的抗原受体，但只有一种受体被激活。 以使它们对病原体做出特异性反应。受体的多样性 在免疫球蛋白（IG）基因中，通过后- 重排体细胞超突变（SHM）和类别转换重组。所有这些过程都涉及 DNA链断裂。重要的是要了解这些途径的调节，这是一部分， 正常的B淋巴细胞发育。过程中的失误已被证明会影响或直接引发 肿瘤发生 这项拟议中的研究将在鲨鱼中进行，鲨鱼是最早的脊椎动物的代表， 基于V（D）J重组的自适应免疫系统。而在小鼠和人类系统中， 3个IG基因座上百个重排基因鲨鱼拥有独特的IG基因组织，基因座多达200个， 每一个包含非常少的（2-4）重组元素。这种组织上的差异， 软骨鱼类和四足动物提供了一个独特的机会， 通过阐明共同的和不同的调控机制来控制IG基因的表达。 具体目标1和3集中在许多微小型（27 IgH，51 IgL）中的活动模式， V（D）J重排。将在基因组和mRNA水平上检查个体B细胞中的IG基因， 为了解导致IG受体最终表达的相互作用因素。因为 初步结果表明，更多的IG基因似乎被激活，而不是严格要求，使一个单一的 功能受体，实验提出1）发现，如果普遍接受的重排连接 在这种情况下，作为调控过程的一部分， DNA重组另外用于使不需要的（例如自身反应性的）IG基因失能。 具体目标2研究鲨鱼B细胞中的重组事件， 胞苷脱氨酶（AID）。AID不仅在编码IG的基因上引起SHM和插入/缺失， 受体，但多个其他IG网站，使缺口和双链断裂引入整个 淋巴细胞活化后的基因组。我们发现来自一种IgH的VDJ可以与C 区域从另一个IgH，有时发生在空间上遥远的 基因.我们建议分离不编码受体的重组产物，包括非- 表示交叉事件的互惠产品，以澄清合作伙伴 基因被靶向重组。了解鲨鱼B细胞的正常过程， 非等位基因之间的交换对于理解疾病状态的发病机理是基本的 由于自身免疫和淋巴肿瘤的易感性异常易位涉及IG基因。

Lymphocytes express a vast repertoire of antigen receptors, but only one species of receptor is allowed per cell in order for them to respond specifically to pathogens. The diversity of the receptors is generated by V(D)J rearrangement and, in immunoglobulin (Ig) genes, further altered by post- rearrangement somatic hypermutation (SHM) and class switch recombination. All these processes involve DNA strand breakage. It is important to understand the regulation of these pathways, which are part of normal B lymphocyte development. Missteps in the process have been shown to influence or directly initiate oncogenesis. The proposed research is to be done in sharks, representatives of the earliest vertebrates with an adaptive immune system based on V(D)J recombination. Whereas in mouse and human systems there are 3 Ig loci with hundreds of rearranging genes, sharks have a unique Ig gene organization of up to 200 loci, each one containing very few (2-4) recombining elements. This organizational disparity between cartilaginous fishes and tetrapods affords a unique opportunity to gain insight into the complex process governing Ig gene expression by elucidating shared and divergent regulatory mechanisms. Specific Aims 1 and 3 focus on patterns of activity among the many miniloci (27 IgH, 51 IgL) during V(D)J rearrangement. The Ig genes in individual B cells will be examined at the genomic and mRNA level in order to understand interplaying factors that bring about the Ig receptor ultimately expressed. Because preliminary results indicate that more Ig genes appear to be activated than strictly required to make a single functional receptor, experiments are proposed to 1) find if the commonly accepted linkage of rearrangement with transcriptional activity is decoupled in this instance, as part of the regulatory process, and 2) establish if DNA recombination is additionally employed to incapacitate unwanted (e.g. auto-reactive) Ig genes. Specific Aim 2 investigates recombination events in shark B cells initiated by activation-induced cytidine deaminase (AID). AID causes SHM and insertions/deletions not only at the genes encoding the Ig receptor but multiple other Ig sites, so that nicks and double-strand breaks are introduced throughout the genome after lymphocyte activation. We discovered that VDJ from one IgH can be expressed with the C region from another IgH, occurring sometimes after unequal crossing-over between the spatially distant genes. We propose isolating the recombination products that do not encode the receptor, including non- expressed reciprocal products of crossing-over events, in order to clarify the conditions under which partner genes are targeted for recombination. Understanding normal processes in shark B cells that lead to genetic exchange between non-allelic genes is fundamental to understanding pathogenesis of disease states such as autoimmunity and predisposal to lymphoid tumors by aberrant translocations involving Ig genes.