MECHANISM OF B LYMPHOCYTE SOMATIC HYPERMUTATION

B淋巴细胞体细胞高突变机制

• 批准号: 2084196
• 负责人: Nancy S. Green
• 金额: $ 9.13万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2084196
• 关键词: B lymphocyte; RNase protection assay; antibody formation; clone cells; electroporation; gel mobility shift assay; gene deletion mutation; gene rearrangement; genetically modified animals; immunoglobulin genes; laboratory mouse; lymphokines; molecular cloning; nucleic acid sequence; point mutation; polymerase chain reaction; protein signal sequence; serology /serodiagnosis; site directed mutagenesis

The long term goal of this physician-scientist grant proposal is to develop the skills required to understand the mechanisms by which B lymphocytes undergo and regulate immunoglobulin (Ig) gene somatic hypermutation. This process allows for generation of greater antibody diversity and development of more effective higher affinity antibodies. Previous attempts to study the molecular mechanisms and detailed organization of this process have been hampered by the lack of an appropriate B cell model for this process. Evidence exists for high frequency mutations of the variable region heavy chain Ig gene of a subclone of 18.81, an Abelson-transformed murine pre-B cell line. During Phase I of this project, in addition to coursework in molecular biology, will be the confirmation that the 18.81 cell line undergoes frequent and spontaneous point mutations in the variable (V) region of its endogenous immunoglobulin gene and that the frequency of V region mutation is greater than constant region mutation. Practical serological and molecular assays will be established to identify mutant genes. Comparisons will be performed of the rate and nature of mutations that occur in transfected and endogenous Ig genes. In Phase II, transfected constructs of an Ig gene will be used to determine DNA sequences in the flanking and/or coding sequences which are required for mutations to occur and to see if specific sequences are targeted for these events. Those sequences shown to be important for this process will be analyzed by site directed mutagenesis and will be used to search for proteins involved in the regulation of this process by gel retardation assay. Their relevance in vivo will be examined using transgenic mice. Genes for these proteins will be cloned for determination of sequence, expression, and in this in vitro system. Determination of the mechanism and regulation of this process would resolve one of the major mechanisms for generating antibody diversity. It would also allow the generation of higher affinity monoclonal antibodies such as those used for anti- neoplastic or infectious processes and for diagnostic purposes. Mutations in the Ig gene are important in the oncogenesis of malignancies such as Burkitt's lymphoma. Improved understanding of this process may lead to novel treatments for these and other lymphoid tumors, and for manipulation of lymphoid cell differentiation.

这项医师-科学家拨款提案的长期目标是开发 了解 B 淋巴细胞的机制所需的技能 经历并调节免疫球蛋白（Ig）基因体细胞超突变。 这 过程允许产生更大的抗体多样性和发展 更有效的更高亲和力的抗体。 之前的学习尝试 该过程的分子机制和详细组织 由于缺乏适合该过程的 B 细胞模型，该过程受到阻碍。 有证据表明重链可变区发生高频突变 18.81亚克隆的链Ig基因，Abelson转化的小鼠前B 细胞系。 在该项目的第一阶段，除了课程作业 分子生物学将证实18.81细胞系 变量 (V) 发生频繁且自发的点突变 其内源性免疫球蛋白基因的区域和V的频率 区域突变大于恒定区突变。 实际的 将建立血清学和分子测定法来鉴定突变体 基因。 将比较突变的速率和性质 发生在转染和内源性 Ig 基因中。 在第二阶段， Ig 基因的转染构建体将用于测定 DNA 侧翼和/或编码序列中的序列是必需的 发生突变并查看特定序列是否针对这些突变 事件。 那些被证明对这个过程很重要的序列将是 通过定点诱变进行分析，并将用于搜索 通过凝胶阻滞参与调节该过程的蛋白质 化验。 将使用转基因小鼠检查它们在体内的相关性。 这些蛋白质的基因将被克隆以确定序列， 表达，以及在这个体外系统中。 机制的确定 对这一过程的监管将解决主要机制之一 用于产生抗体多样性。 它还将允许生成 更高亲和力的单克隆抗体，例如用于抗- 肿瘤或感染过程以及用于诊断目的。 突变 Ig 基因中的 Ig 基因在恶性肿瘤的发生中很重要，例如 伯基特淋巴瘤。 加深对这一过程的理解可能会导致 针对这些和其他淋巴肿瘤以及操纵的新疗法 淋巴细胞分化。