GENERATION OF ANTIBODY DIVERSITY

抗体多样性的产生

• 批准号: 3301931
• 负责人: PATRICIA J GEARHART
• 金额: $ 29.66万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301931
• 关键词: B lymphocyte; Bordetella pertussis; DNA directed DNA polymerase; DNA repair; T cell receptor; antibody formation; cell free system; cell fusion; endonuclease; enzyme mechanism; exonuclease; gene deletion mutation; gene expression; gene mutation; gene rearrangement; genetic manipulation; genetic mapping; genetic regulatory element; genetic techniques; genetic transcription; genetically modified animals; hemocyanin; hybridomas; hydroxylamine; immunoglobulin genes; laboratory mouse; molecular cloning; nucleic acid sequence; osmium; polymerase chain reaction; protein purification; reporter genes; technology /technique development; tissue /cell culture; tissue mosaicism; transfer RNA

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.

小鼠免疫球蛋白基因的体细胞超突变，发生在 每代每bp 10-3的速率，定位于DNA的2kb区域 围绕并包括重排变量（V）、多样性（D），以及 连接（J）编码重链和轻链可变区的基因区段。 提出VDJ基因的独特序列和结构， 侧翼区编码的信息，导致突变的易错 修复. 两个主要目标是鉴定顺式DNA序列， 靶向VDJ基因突变，并鉴定B细胞中的酶， 参与了容易出错的修复 由于突变几乎是在 每一个被抗原刺激的B淋巴细胞， 了解这个机制很重要。 第一个目标是发展 产生和检测质粒载体中突变的测定和系统 含有VDJ基因。 将开发两种检测方法，以快速扫描 突变 第一种是基因检测， 报告基因位于VDJ基因旁边。 tRNA的完整性 通过细菌菌落的颜色监测分子。 第二个是 通过切割错配的 异源双链体。 突变的模板通过PCR扩增，并且错配 核苷酸被四氧化锇和羟胺化学修饰。 对于这两种测定，突变将通过测序来确认。 三个系统 将尝试在质粒载体上产生突变：（i）SP2/0 将含有质粒的杂交瘤细胞与抗原活化的B融合 细胞，并在体外发生突变;（ii）质粒将用于使 将被免疫的转基因小鼠，和（ii）质粒将被 重组到多能干细胞中的同源免疫球蛋白位点 细胞，然后将其发育成嵌合小鼠并进行免疫。 的 第二个目的是鉴定VDJ基因周围的顺式序列， 参与产生突变。 使用该测定和系统， 突变频率最高时，将构建质粒， VDJ基因周围2-kb区域的缺失，以及质粒 将进行突变检测 当一个关键序列被删除，突变 将被废除。 第三个目的是研究躯体化的机制， 通过鉴定B细胞核提取物中的酶活性进行突变。 细胞核提取物将从处于不同发育阶段的B细胞制备。 分化并测定核酸内切酶和核酸外切酶活性。 特异性将在两个水平之一显示：（i）细胞特异性，其中 B细胞提取物含有酶，但不含其他细胞，或（ii）底物 特异性，其中VDJ基因突变，而不是其他基因。 外切酶 活性将通过引物延伸来测定，以定位切口具有 发生了。 外切核酸酶活性将通过降解 放射性标记的DNA 蛋白质将在体外进行纯化和测试， 突变的机制。