RUI: Anaylsis of Somatic Mutation in Immunoglobulin V Genes Using a Reporter Gene Assay

9419042免疫球蛋白可变(V)基因的Pollock体细胞突变在抗体的产生多样性中起着重要作用。正在开发一种检测和分离体内和体外体细胞突变的细胞的方法。该分析基于编码融合蛋白的DNA构建，该融合蛋白由重排的V基因组成，包含一个终止密码子，与LacZ基因相连。V基因是LacZ基因的5‘端，其终止密码子因此阻止了LacZ基因的翻译，如果体细胞突变逆转了终止密码子，则整个融合蛋白被翻译并表达LacZ。LacZ的表达可以通过组织化学或荧光分析很容易地检测到。将此构建物导入细胞系，分离出表达LacZ的突变型细胞系。含有这种结构的转基因小鼠正在制造中。利用转基因小鼠进一步研究体细胞突变并解决6个基本问题：1)在哪里可以检测到体细胞突变；2)免疫后什么时候出现体细胞突变；3)体细胞突变最早发生在个体发育期间的什么时候；4)哪些生发中心细胞显示出活跃的体细胞突变；5)体细胞突变的表型是什么；以及6)发生了什么类型的突变。%虽然哺乳动物的免疫系统如何产生大量的抗体多样性是已知的，但基因组DNA重排并不是全部答案。免疫球蛋白可变基因的体细胞突变在产生这种多样性中起着重要作用。这一努力将要求对六个基本问题进行调查。在哪里可以检测到体细胞突变？在个体发育过程中，体细胞突变最早发生在什么时候？哪些生发中心细胞显示活跃的体细胞突变？正在经历体细胞突变的细胞的表型是什么？会发生哪些类型的突变？这些问题的答案既有基本利益，也有现实利益。***

9419042 Pollock Somatic mutation of immunoglobulin variable (v) genes plays an important role in the generating antibody diversity. An assay is being developed to detect and isolate cells undergoing somatic mutation in vivo and in vitro. The assay is based on a DNA construct encoding a fusion protein composed of a rearranged V gene, containing a stop codon, linked to a lacZ gene. The V gene is 5' of the lacZ; its stop codon therefore prevents transltion of the lacZ gene, If somatic mutation revers the stop codon, the entire fusion protein is translated and lacZ expressed. LacZ expression can be readily detected by histochemical or fluorescence assays. This construct has been put into cell lines and a mutant cell line expressing lacZ isolated. Transgenic mice containing this construct are being made. Experiments are proposed to further study mutation in vitro and to address six basic questions using the transgenic mice: 1) where can somatic mutation be detected; 2) when is somatic mutation seen after immunization; 3) when does somatic mutation first occur during ontogeny; 4) which germinal center cells show active somatic mutation; 5) what is the phenotype of cells undergoing somatic mutation; and 6) what types of mutations occur. %%% Although it is know how the mammalian immune system generates much of the antibody diversity that is seen, genomic DNA rearrangement is not the whole answer. Somatic mutation of immunoglobulin variable genes plays an important role in generating such diversity. An investigation into six basic questions will be asked by this effort. Where can somatic mutation be detected ? When does somatic mutation first occur during ontogeny? Which germinal center cells show active somatic mutation? What is the phenotype of cells undergoing somatic mutation? What types of mutations occur? The answers to those questions have both basic and practical interest. ***