ANTIBODY VARIABLE GENES: DEVELOPMENT AND DIVERSITY

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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)