BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

描述：(改编自申请人的摘要)编码这两个基因 免疫系统中关键类型的抗原识别分子 免疫球蛋白(Ig)和T细胞受体(TCR)在 一种新的、高度调控的位点特异性DNA对淋巴细胞的发育 复合反应称为V(D)J复合。组合式 基因片段的连接允许免疫系统近乎无限地编码 抗原识别能力，只需适度的基因投资。这个 然而，发育中的淋巴细胞面临着调节这一点的问题。 反应以产生功能性Ig或TCR，同时避免 体细胞重组所固有的基因组不稳定性。几个新的 已经开发了一些技术来研究V(D)J的细节 重组反应途径及其调控的分子机制 这种反应。 研究人员设计了一系列基于聚合酶链式反应的分析方法，使 V(D)J复合反应中间体的检测 特别是断裂的DNA分子。这些检测将被用来确定 断裂DNA编码末端的精确结构，它们在 正在进行基因重排的细胞的核，这些 末端，以及与末端结合的蛋白质的身份。这个 认为V(D)J重组异常参与了骨肉瘤的发病机制 白血病相关染色体易位将通过寻找 隐蔽重组信号序列(RSS)处的DNA断裂 原癌基因。最近，美国国立卫生研究院的研究人员首次报道了 可特异性识别和切割重排的体外系统 基因。这个系统将被用来解决如何确定目标的问题 重组酶是通过进行体外切割试验来调节的 从各种淋巴前体中分离出的核。染色质的想法 结构决定了重组酶对靶基因座的选择 测试过。最后，确定最低活跃度的“核心”域 重组酶基因RAG-1和RAG-2可以挽救B细胞的发育 RAG-I或RAG-2缺陷转基因小鼠，表达这些蛋白的小鼠 都会被制造出来。最后，研究人员将筛查淋巴组织中的蛋白质 与碎屑中不必要的区域相互作用的前体。

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.