ANTIBODY VARIABLE GENES: DEVELOPMENT AND DIVERSITY

这项研究的两个目标是：(1)确定是否存在 抗体可变基因表达的发育顺序和(2) 确定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 在BALB/c小鼠中有超过100V的重链(VH)基因。 免疫球蛋白重链在11天左右开始表达。 胚胎发生。我们将首先确定VH基因是否表达 在个体发育过程中非随机的。含有重排VH基因的前B细胞 从第13天到第17天，胎儿肝脏将与Abelson一起转化 病毒。来自细胞系的mRNA将与各种VH杂交 对应于不同家族的探头。结果将表明，如果 VH基因重排有一定的发育规律。 其次，该模式将与VH基因的染色体图谱进行比较。 VH基因的表达和组织之间的关系可能 提出了这个多基因家族的发育控制机制。 这项工作的另一个结果是在前B细胞中出现了新的VH基因 将被发现、描述和绘制，这将扩展我们的 VH基因家族知识。一种特定的体细胞突变机制 只突变重排的V基因，而不突变相邻的恒定基因 以10-2的极高频率引入点突变。我们会 尝试识别影响V基因的核苷酸序列 突变。将开发一种检测突变的方法，使用V基因检测 牛乳头瘤病毒载体上的kappa轻链(Vk167) 在真核细胞中稳定复制。该载体将被转化为 抗原刺激的B细胞，后来恢复以确定突变是否 发生了。将尝试三种方法来开发这种分析方法：(1) 邻接的、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西林上生长；(2)遗传 具有无义突变的Vk167缺陷基因的逆转 编码区将允许表达抗磷胆碱抗体；以及 (3)Vk167基因序列测定。一旦开发出一种化验方法， Vk167基因周围的DNA将被删除，并进行突变检测。什么时候 假定的突变子序列被删除，不应该发生突变。(磅)

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)