BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

描述：（改编自申请人的摘要） 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 淋巴细胞发育通过一种新的，高度调控的位点特异性DNA 这种复合反应称为V（D）J复合。 的组合 基因片段的连接使免疫系统能够编码几乎无限的 抗原识别能力，只有适度的遗传投资。 的 然而，发育中的淋巴细胞面临着调节这种调节的问题。 产生功能性IG或TCR的反应，同时避免 体细胞重组中固有的基因组不稳定性。 几个新 研究V（D）J细节的技术已经发展起来 重组反应途径和调节的分子机制 这种反应。 研究人员设计了一系列基于PCR的检测方法， 检测V（D）J重组反应中间体， 特别是断裂的DNA分子 这些试验将用于确定 断裂DNA编码末端的精确结构、它们在其中的位置 细胞核进行基因重排，这些可及性 末端，以及与它们结合的蛋白质的身份。 的 假设异常V（D）J重组参与了 白血病相关的染色体易位将通过寻找 DNA在隐蔽重组信号序列（RSS）处断裂， 原癌基因 最近，美国国立卫生研究院的研究人员报告了第一个 能特异性识别和切割重排的体外系统 基因. 这一系统将用于解决如何确定目标的问题， 重组酶通过体外切割试验进行调节， 从各种淋巴前体分离的细胞核。 染色质 结构决定了重组酶对靶基因座的选择， 测试. 最后，为了确定最低活跃“核心”域是否 重组酶基因RAG-1和RAG-2可以拯救B细胞发育， RAG-I或RAG-2缺陷型转基因小鼠，表达这些蛋白质的小鼠 将被制造。 最后，研究者将筛选淋巴细胞中的蛋白质， 与RAG的非必需结构域相互作用的前体。

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.