ANTIBODY VARIABLE GENES: DEVELOPMENT AND DIVERSITY

抗体可变基因：发展和多样性

• 批准号: 3171881
• 负责人: PATRICIA J GEARHART
• 金额: $ 13.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-08-01 至 1987-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3171881
• 关键词: Abelson leukemia virus; B lymphocyte; antibody specificity; clone cells; endonuclease; gene expression; genetic mapping; immunoglobulins; leukocyte activation /transformation; monoclonal antibody; myeloma globulin; nucleic acid sequence; phosphorylcholine; radioimmunoassay

The two goals of the research are: (1) to determine if there is a developmental order to antibody variable gene expression and (2) to identify nucleotide sequences around the variable gene that influence somatic mutation. In the immunoglobulin variable (V) gene family, there are over 100 V genes for the heavy chain (VH) in BALB/c mice. Immunoglobulin heavy chains begin to be expressed around day 11 of embryogenesis. We will first determine if VH genes are expressed nonrandomly during ontogeny. Pre-B cells containing rearranged VH genes from day 13 through day 17 in fetal liver will be transformed with Abelson virus. mRNA from the cell lines will be hybridized with a variety of VH probes corresponding to different families. The results will indicate if there is a developmental pattern to the rearrangement of VH genes. Second, the pattern will be compared to the chromosomal map of VH genes. The relationship between expression and organization of VH genes may suggest mechanisms for the developmental control of this multigene family. Another outcome of this work is that new VH genes arising in pre-B cells will be discovered, characterized, and mapped, which will expand our knowledge of VH gene families. A somatic mutation mechanism specificaIly mutates only rearranged V genes and not adjacent constant genes and introduces point mutations at a very high frequency of 10-2. We will attempt to identify nucleotide sequences around V genes that influence mutation. An assay to detect mutation will be developed using a V gene for the kappa light chain (Vk167) on a bovine papillomavirus vector that stably replicates in eukaryotic cells. The vector will be transfected into antigen-stimulated B cells and later recovered to determine if mutation has occurred. Three approaches to develop this assay will be tried: (1) genetic reversion of an adjacent, defective betalactamase gene that has a nonsense mutation will permit bacterial growth on ampicillin; (2) genetic reversion of a defective Vk167 gene that has a nonsense mutation in the coding region will allow expression of anti-phosphorylcholine antibody; and (3) sequencing of the Vk167 gene. Once an assay is developed, regions of DNA around the Vk167 gene will be deleted and tested for mutation. When the presumed mutator sequence is deleted, mutation should not occur. (LB)

研究的两个目标是：（1）确定是否存在 抗体可变基因表达的发育顺序和（2） 识别影响可变基因周围的核苷酸序列 体细胞突变。 在免疫球蛋白可变（V）基因家族中，有 BALB/c 小鼠中有超过 100 个重链 (V H ) V 基因。 免疫球蛋白重链在第11天左右开始表达 胚胎发生。 我们首先确定V H 基因是否表达 在个体发育过程中非随机。 含有重排 V H 基因的前 B 细胞 从第 13 天到第 17 天，胎儿肝脏将通过 Abelson 进行转化 病毒。 来自细胞系的 mRNA 将与多种 VH 杂交 探针对应不同的家族。 结果将表明如果 V H 基因的重排有一个发育模式。 其次，该模式将与 V H 基因的染色体图谱进行比较。 V H 基因的表达和组织之间的关系可能 提出了该多基因家族发育控制的机制。 这项工作的另一个成果是前 B 细胞中出现了新的 VH 基因 将被发现、表征和绘制地图，这将扩展我们的 V H 基因家族的知识。 体细胞突变机制 仅突变重排的 V 基因，而不突变相邻的恒定基因，并且 以 10 -2 的极高频率引入点突变。 我们将 尝试鉴定V基因周围影响的核苷酸序列 突变。 将开发一种使用 V 基因检测突变的方法 牛乳头瘤病毒载体上的 kappa 轻链 (V k167 ) 在真核细胞中稳定复制。 该载体将被转染到 抗原刺激的 B 细胞，然后恢复以确定是否发生突变 发生。 将尝试三种方法来开发该测定：(1) 相邻的有缺陷的β内酰胺酶基因的遗传逆转 无义突变将允许细菌在氨苄青霉素上生长； (2)遗传 有缺陷的 V k167 基因的回复，该基因在 编码区将允许抗磷酸胆碱抗体的表达；和 (3)V k167基因测序。 一旦开发出一种检测方法，区域 V k167 基因周围的 DNA 将被删除并测试突变。 什么时候 假定的突变序列被删除，突变不应发生。 （磅）