ANTIBODY VARIABLE GENES: DEVELOPMENT AND DIVERSITY

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， 重链（V）有超过100个V基因H）。 免疫球蛋白重链开始表达大约在免疫球蛋白重链的第11天。 胚胎发生 我们先来看看VH 基因被表达 在个体发育过程中是非随机的 含有重排V的前B细胞H 基因 从第13天到第17天的胎肝将用Abelson转化 病毒 来自细胞系的mRNA将与多种VH 探针对应不同的家庭。 结果将表明，如果 V的重排有一个发展模式H 基因. 第二，将该模式与V染色体图谱进行比较，H 基因. V的表达与组织的关系H 基因可能 提出了这个多基因家族的发育控制机制。 这项工作的另一个成果是，新的VH 前B细胞中产生的基因 将被发现，表征和映射，这将扩大我们的 知识VH 基因家族 一种体细胞突变机制 仅突变重排的V基因而不突变相邻的恒定基因， 以非常高的频率引入点突变-2. 我们将 试图确定V基因周围的核苷酸序列， 突变 将使用V基因开发检测突变的测定法， κ轻链（VK167）在牛乳头瘤病毒载体上， 在真核细胞中稳定复制。 将载体转染入 抗原刺激的B细胞，随后回收，以确定突变是否 发生了。 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的回复K167 一个无义突变的基因， 编码区将允许抗磷酸胆碱抗体的表达;和 (3)V的排序K167 基因 一旦开发了测定， V周围的DNAK167 基因将被删除并进行突变测试。 当 假定的突变子序列被删除，突变不应该发生。（LB）

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)