Recently molecular approaches have demonstrated mechanisms involved in generation of diversity within immunoglobulin genes. These include the description of rearrangement of germ-line segments (variable-diversity-joining, V/H-D-J/H for the heavy chain and variable-joining, V/L-J/L for the light chain), recombinational diversity (sequences added in the recombination process), combinatorial diversity (use of different heavy and light chains) and somatic mutation processes. These methods diversification operate on germ-line sequences which, to date, are not described. This proposal focuses on the structural characterization of a single V/H gene family and functional studies of usage frequency of V/H sequences. The V/H 7183 gene family was chosen for study for several reasons. This multigene family has been mapped to be the most proximal to the D-J/H sequences and appears to be preferentially rearranged in B cells derived from fatal tissues. The gene family is of moderate size and appears to be remarkably conserved in all chordates and several sequences within the gene family display striking regions of homology. To gain insight into mechanisms that could be operating to maintain such homology, gene segments of the V/H 7183 gene family will be cloned and rapidly sequenced using synthetic oligonucleotide strategies. Alleles of functional genes, a pseudogene and noncoding flanking regions from the BALB/c mouse will be compared in C57BL/6 and A.CA mouse strains. Characterization of the type of mutations (clustered or random) and numbers of substitutions that have accumulated between allelic segments will give information into rates of sequence divergence and suggest possible genetic mechanisms that may operate within this gene family Functional usage of V/H gene families and genes within a family will be approached in two ways: (1) RNA obtained from LPS stimulated hybridomas derived from several sources, fetal liver, neonatal spleens and adult spleens, will be characterized in a dot blot analysis with eight different V/H gene probes. Usage of specific genes within the V/H 7183 gene family will be achieved by hybridization of RNA from the hybridomas with gene specific synthetic oligonucleotides; (2) LPS stimulated B cells obtained from the same sources described above will be grown as colonies in filter discs, lysed and fixed. The RNA can be hybridized with the same V/H gene probes. Similarly, gene specific synthetic oligonucleotide can be used to differentiate individual gene usage within a gene family.
