IMMUNOGLOBULIN AND T CELL RECEPTOR GENE ASSEMBLY

免疫球蛋白和 T 细胞受体基因组装

• 批准号: 3147642
• 负责人: David G. Schatz
• 金额: $ 7.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3147642
• 关键词: DNA topoisomerases; T cell receptor; chimeric proteins; endonuclease; enzyme activity; exonuclease; gene expression; gene rearrangement; glutathione transferase; hamsters; immunoglobulin genes; laboratory mouse; laboratory rabbit; laboratory rat; ligase; molecular cloning; monoclonal antibody; protein purification; protein structure function; recombinase; tissue /cell culture

Cells of the immune system act in concert to protect against infectious agents and transformed cells. At the heart of this protective system are the clonotypic antigen receptor molecules found on B and T lymphocytes, the immunoglobulin (Ig) and the T cell receptor (TCR). The millions of different genes needed to encode these receptors are assembled from component gene segments by a site-specific recombination process known as V(D)J recombination. Aberrant V(D)J recombination has been linked to human hematopoietic malignancy, and defects in or deregulation of the recombination process could lead to immunodeficiency or autoimmunity. To understand the mechanisms by which V(D)J recombination causes disease, the process needs to be understood at the molecular level. The primary objective of the research described in this application is to identify the enzymatic machinery that carries out V(D)J recombination and determine the role of each enzymatic component in the reaction. Two recombination activating genes, RAG-1 and RAG-2, have been isolated and demonstrated to be necessary and sufficient to activate the V(D)J recombination machinery in non-lymphoid cells. These genes likely encode the critical lymphoid-specific components of the recombination enzyme. They offer unique reagents with which to study the biochemistry of V(D)J recombination. The cloned RAG-1 and RAG-2 cDNAs will be used to direct the expression of the RAG-1 and RAG-2 proteins (and small portions of the proteins) in bacterial and mammalian tissue culture cells. The proteins will be purified by taking advantage of a variety of affinity purification schemes, and used as immunogens to generate polyclonal antisera and monoclonal antibodies specific for the RAG proteins. These immunological reagents will be used to determine the in vivo pattern of expression of the RAG-1 and RAG-2 proteins, both in lymphoid organs and tissues, and in the central nervous system (where the RAG-1 mRNA transcript is found). The antibodies will also be used to identify other components of the V(D)J recombinational machinery by virtue of their interactions with the RAG proteins. The biological activity of the purified RAG proteins will be assayed by introducing the proteins directly into tissue culture cells. The proteins will also be analyzed in detail for their ability to bind DNA (particularly to sequences known to be required for V(D)J recombination) and for their enzymatic activities, particularly as topoisomerases, endo- and exonucleases, and ligases. The information gained in these studies will be used to develop an in vitro V(D)J recombination assay with which the enzymatic mechanism of the reaction can be determined. Identification of the components of the enzyme and an understanding of the role each plays in the recombination reaction should provide insights into the mechanisms by which defects in the reaction lead to human disease.

免疫系统的细胞共同起作用可预防感染性 代理和转化的细胞。 这个保护系统的核心是 在B和T淋巴细胞上发现的clonotypic抗原受体分子， 免疫球蛋白（IG）和T细胞受体（TCR）。 数百万 编码这些受体所需的不同基因是从中组装的 分量基因段通过特定于地点的重组过程称为 V（d）J重组。 异常V（d）J重组与人类有关 造血恶性肿瘤，并在 重组过程可能导致免疫缺陷或自身免疫性。 到 了解V（d）J重组引起疾病的机制， 需要在分子水平上了解过程。 本应用程序中描述的研究的主要目的是 确定执行V（d）J重组和 确定每个酶成分在反应中的作用。 二 重组激活基因RAG-1和RAG-2已被分离，并且 证明是必要的，足以激活V（d）J 非淋巴细胞中的重组机制。 这些基因可能编码 重组酶的临界淋巴特异性成分。 他们提供了研究V（d）J的生物化学的独特试剂 重组。 克隆的RAG-1和RAG-2 cDNA将用于指导表达 RAG-1和RAG-2蛋白（以及蛋白的一小部分） 细菌和哺乳动物组织培养细胞。 蛋白质将是 通过利用各种亲和力纯化方案纯化， 并用作免疫原以产生多克隆抗血清和单克隆 对抹布蛋白的特异性抗体。 这些免疫学试剂 将用于确定rag-1的体内表达模式 和RAG-2蛋白，包括淋巴机构和组织，以及中央 神经系统（发现RAG-1 mRNA转录本）。 抗体 还将用于识别V（d）J重组的其他组件 机械凭借其与抹布蛋白的相互作用。 纯化的抹布蛋白的生物学活性将通过 将蛋白质直接引入组织培养细胞。 蛋白质 还将详细分析其结合DNA的能力（尤其是 对于V（d）J重组所需的序列）及其 酶活性，特别是作为拓扑异构酶，内部和 外核酸和连接酶。 这些研究中获得的信息将是 用于开发体外V（d）J重组测定法 可以确定反应的酶促机制。 识别 酶的成分以及对每个扮演的作用的理解 重组反应应通过 反应中的缺陷导致人类疾病。