GENERATION OF ANTIBODY DIVERSITY

小鼠免疫球蛋白基因的体细胞高度突变，发生在 每代10-3个碱基对，定位于DNA的2kb区域 围绕并包括重排变量(V)、多样性(D)和 连接(J)编码重链和轻链可变区的基因片段。 VDJ基因独特的序列和结构被认为是 侧翼区域对信息进行编码，以导致容易出错的突变 修理。这两个主要目标是识别顺式DNA序列， VDJ基因的靶向突变，并鉴定B细胞中可 涉及到容易出错的修复。由于突变在近几年被激活 每一个被抗原刺激的B淋巴细胞，它都是至关重要的 了解这一机制的重要性。第一个目标是发展 在质粒载体中产生和检测突变的分析和系统 含有VDJ基因。将开发两种检测方法来快速扫描 突变。拳头是一种对tRNA突变进行评分的遗传分析方法。 报道基因被放置在VDJ基因旁边。TRNA的完整性 通过细菌菌落的颜色来监测分子。第二个是一个 通过错配的裂解来检测突变的化学方法 异源双链。突变的模板通过聚合酶链式反应进行扩增，并且不匹配 核苷酸是由四氧化三和羟胺进行化学修饰的。 对于这两种检测，突变将通过测序得到确认。三大体系 将尝试在质粒载体上产生突变：(I)SP2/0 含有质粒的杂交瘤细胞将与抗原激活的B细胞融合 细胞，并在体外发生突变；(Ii)质粒将被用来制造 将被免疫的转基因小鼠，以及(Ii)将被 重组到多能干细胞中的同源免疫球蛋白部位 细胞，然后这些细胞将被发育成嵌合小鼠并免疫。这个 第二个目标是确定VDJ基因周围的顺式序列 参与了突变的产生。使用给出的分析和系统 最高频率的突变，构建的质粒将包含 VDJ基因周围2-kb区域的缺失，以及质粒 将接受突变检测。当一个关键序列被删除时，突变 将被废除。第三个目的是研究体细胞分裂的机制。 通过鉴定B细胞核抽提物中的酶活性进行突变。 将从处于不同阶段的B细胞制备核提取液 并进行核酸内切酶和核酸外切酶活性测定。 特异性将显示在以下两个水平之一：(I)细胞特异性，其中 B细胞提取物含有这种酶，但不含其他细胞，或(Ii)底物 特异性，VDJ基因突变的地方，而不是其他基因。核酸外切酶 将通过引物延伸来分析活性，以绘制出有NICK的位置 发生了。核酸外切酶的活性将通过降解 放射性标记的DNA。蛋白质将被提纯并在体外进行测试以进行研究 突变的机制。

Somatic hypermutation in murine immunoglobulin genes, which occurs at a rate of 10-3 per bp per generation, is localized to a 2-kb region of DNA surrounding and including rearrange variable (V), diversity (D), and joining (J) gene segments encoding heavy and light chain variable regions. It is proposed that the unique sequence and structure of the VDJ gene and flanking regions encodes the information to cause mutation by error-prone repair. The tow major objectives are to identify cis DNA sequences that target mutation to the VDJ gene, and to identify enzymes in B cells that are involved in error-prone repair. Since mutation is activated in nearly every B lymphocyte that is stimulated with antigen, it is of paramount importance to understand this mechanism. The first aim is to develop assays and systems that generate and detect mutation in plasmid vectors containing VDJ genes. Two assays will be developed to rapidly scan for mutation. The fist is a genetic assay that scores for mutation in a tRNA reporter gene placed next to the VDJ gene. The integrity of the tRNA molecule is monitored by color of bacterial colonies. The second is a chemical assay which detects mutation by cleavage of mismatched heteroduplexes. Mutated templates are amplified by PCR, and mismatched nucleotides are chemically-modified by osmium tetroxide and hydroxylamine. For both assays, mutations will be confirmed by sequencing. Three systems will be tried to generate mutation on the plasmid vectors: (i) SP2/0 hybridoma cells containing plasmids will be fused to antigen-activated B cells, and mutation will occur in vitro; (ii) plasmids will be used to make transgenic mice which will be immunized, and (ii) plasmids will be recombined into the homologous immunoglobulin site in pluri-potent stem cells, which will then be developed into chimeric mice and immunized. The second aim is to identify cis sequences around the VDJ gene that are involved in generating mutation. Using the assay and system which gives the highest frequency of mutation, plasmids will be constructed to contain deletions of the 2-kb region surrounding the VDJ gene, and the plasmids will be tested for mutation. When a critical sequence is deleted, mutation will be abolished. The third aim is to examine the mechanism of somatic mutation by identifying enzymatic activities in B-cell nuclear extracts. Nuclear extracts will be prepared from B cells in various stages of differentiation and assayed for endonuclease and exonuclease activities. Specificity will be shown at one of two levels: (i) cell specificity, where B-cell extracts contain the enzyme, but not other cells, or (ii) substrate specificity, where VDJ genes mutate, but not other genes. Exonuclease activity will assayed by primer extension to map the sites where nicks have occurred. Exonuclease activity will be assayed by degradation of radiolabeled DNA. Proteins will be purified and tested in vitro to study the mechanism of mutation.