BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

V(D)J 重组的生物化学和调控

• 批准号: 2672836
• 负责人: Mark S. Schlissel
• 金额: $ 26.09万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672836
• 关键词: B lymphocyte; DNA damage; cell differentiation; gene rearrangement; genetic library; genetically modified animals; immunoglobulin genes; laboratory mouse; polymerase chain reaction; protooncogene; recombinase

DESCRIPTION: (Adapted from the applicant's abstract) Genes encoding the two critical types of antigen recognition molecules in the immune system immunoglobulin (Ig) and T cell receptor (TCR), are assembled during lymphocyte development by a novel, highly regulated site-specific DNA recombination reaction known as V(D)J recombination. The combinatorial joining of gene segments allows the immune system to encode near limitless antigen recognition capability with only a modest genetic investment. The developing lymphocyte is faced with the problem, however, of regulating this reaction to produce functional Ig or TCR while avoiding the consequences of the genomic instability inherent in somatic recombination. Several new techniques have been developed to study the details of the V(D)J recombination reaction pathway and the molecular mechanisms which regulate this reaction. The investigator has devised a series of PCR-based assays which allow detection of V(D)J recombination reaction intermediates consisting of specifically broken DNA molecules. These assays will be used to determine the precise structure of broken DNA coding ends, their location within nuclei of cells undergoing gene rearrangement, the accessibility of these ends, and the identities of proteins which are bound to them. The hypothesis that aberrant V(D)J recombination is involved in the etiology of leukemia associated chromosomal translocation will be tested by looking for DNA breaks at cryptic recombination signal sequences (RSSs) in proto-oncogenes. Recently, researchers at the NIH reported the first in vitro system capable of specific recognition and cleavage of rearranging genes. This system will be used to address the question of how targeting of the recombinase is regulated by performing in vitro cleavage assays on nuclei isolated from various lymphoid precursors. The idea that chromatin structure dictates the choice of target loci by the recombinase will also be tested. Finally, to determine whether the minimally active "core" domains of recombinase genes RAG-1 and RAG-2 can rescue B-cell development in either RAG-I or RAG-2 deficient transgenic mice, mice expressing these proteins will be made. Lastly, the investigator will screen for proteins in lymphoid precursors which interact with the unessential domains of the RAGs.

描述：（改编自申请人的摘要）编码这两个的基因 免疫系统中抗原识别分子的关键类型 免疫球蛋白（IG）和T细胞受体（TCR）在期间组装 通过一种新型的，高度调节的位点特异性DNA的淋巴细胞发展 重组反应称为V（d）J重组。 组合 基因段的连接允许免疫系统编码接近无限 抗原识别能力仅具有适中的遗传投资。 这 但是，发展淋巴细胞面临调节的问题 产生功能Ig或TCR的反应，同时避免 体细胞重组固有的基因组不稳定性。 几个新 已经开发了研究V（d）J的细节的技术 重组反应途径和调节的分子机制 这个反应。 调查人员设计了一系列基于PCR的测定法 V（d）J重组反应中间体的检测 特异性破裂的DNA分子。 这些测定将用于确定 断裂的DNA编码结束的确切结构结束，它们的位置 经过基因重排的细胞核，这些细胞的可及性 结束，以及与之绑定的蛋白质的身份。 这 假设异常V（d）J重组与病因有关 白血病相关的染色体易位将通过寻找 DNA在神秘重组信号序列（RSS）中断裂 原始基因。 最近，NIH的研究人员报告了第一项 体外系统，能够具体识别和重新排列的裂解 基因。 该系统将用于解决针对目标的问题 通过对体外切割测定法调节重组酶 从各种淋巴前体分离的细胞核。 染色质的想法 结构决定重组酶选择目标基因座的选择也将是 测试。 最后，确定最小活动的“核心”域是否 重组酶基因rag-1和rag-2可以在任何一个中挽救B细胞的发育 RAG-I或RAG-2缺乏转基因小鼠，表达这些蛋白质的小鼠 会做。 最后，研究者将筛选淋巴样蛋白 与抹布的不必要域相互作用的前体。