MECHANISM OF B LYMPHOCYTE SOMATIC HYPERMUTATION

这项医生-科学家资助提案的长期目标是开发 理解B淋巴细胞的机制所需的技能 经历和调节免疫球蛋白(Ig)基因体细胞超突变。这 该过程允许产生更大的抗体多样性和发展 更有效、更高亲和力的抗体。以前的研究尝试 这一过程的分子机制和详细组织有 由于缺乏适用于这一过程的B细胞模型而受到阻碍。 存在可变区Heavy高频突变的证据 Abelson转化的小鼠Pre-B亚克隆18.81的链Ig基因 细胞系。在这个项目的第一阶段，除了 分子生物学，将证实18.81细胞系 在变量(V)中经历频繁和自发的点突变 其内源性免疫球蛋白基因的区域和V基因的频率 区域突变大于恒定区域突变。实用 将建立血清学和分子检测来鉴定突变 基因。将对突变的速度和性质进行比较 发生在转基因和内源性免疫球蛋白基因中。在第二阶段， 转基因的免疫球蛋白基因将被用来检测DNA 所需的侧翼序列和/或编码序列中的序列 发生突变，并查看特定序列是否针对这些 事件。那些被证明对这一过程很重要的序列将是 通过定点突变进行分析，并将用于搜索 通过凝胶滞留调节这一过程的蛋白质 化验。它们在体内的相关性将使用转基因小鼠进行检验。 这些蛋白质的基因将被克隆以确定序列， 表达，并在这个体外系统中。机理的确定 而对这一过程的监管将解决一个主要机制 以产生抗体多样性。它还将允许产生 亲和力更高的单抗，如用于抗人 肿瘤性或感染性过程和诊断目的。突变 在恶性肿瘤的发生过程中起着重要的作用 伯基特淋巴瘤。对这一过程的更好理解可能会导致 这些和其他淋巴样肿瘤的新治疗方法和操作 淋巴样细胞分化。

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.