GENERATION OF ANTIBODY DIVERSITY

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Somatic hypermutation in murine immunoglobulin genes, which occurs at a rate of 10-3 per bp per generation, is localized to a 2-kb region of DNA surrounding and including rearrange variable (V), diversity (D), and joining (J) gene segments encoding heavy and light chain variable regions. It is proposed that the unique sequence and structure of the VDJ gene and flanking regions encodes the information to cause mutation by error-prone repair. The tow major objectives are to identify cis DNA sequences that target mutation to the VDJ gene, and to identify enzymes in B cells that are involved in error-prone repair. Since mutation is activated in nearly every B lymphocyte that is stimulated with antigen, it is of paramount importance to understand this mechanism. The first aim is to develop assays and systems that generate and detect mutation in plasmid vectors containing VDJ genes. Two assays will be developed to rapidly scan for mutation. The fist is a genetic assay that scores for mutation in a tRNA reporter gene placed next to the VDJ gene. The integrity of the tRNA molecule is monitored by color of bacterial colonies. The second is a chemical assay which detects mutation by cleavage of mismatched heteroduplexes. Mutated templates are amplified by PCR, and mismatched nucleotides are chemically-modified by osmium tetroxide and hydroxylamine. For both assays, mutations will be confirmed by sequencing. Three systems will be tried to generate mutation on the plasmid vectors: (i) SP2/0 hybridoma cells containing plasmids will be fused to antigen-activated B cells, and mutation will occur in vitro; (ii) plasmids will be used to make transgenic mice which will be immunized, and (ii) plasmids will be recombined into the homologous immunoglobulin site in pluri-potent stem cells, which will then be developed into chimeric mice and immunized. The second aim is to identify cis sequences around the VDJ gene that are involved in generating mutation. Using the assay and system which gives the highest frequency of mutation, plasmids will be constructed to contain deletions of the 2-kb region surrounding the VDJ gene, and the plasmids will be tested for mutation. When a critical sequence is deleted, mutation will be abolished. The third aim is to examine the mechanism of somatic mutation by identifying enzymatic activities in B-cell nuclear extracts. Nuclear extracts will be prepared from B cells in various stages of differentiation and assayed for endonuclease and exonuclease activities. Specificity will be shown at one of two levels: (i) cell specificity, where B-cell extracts contain the enzyme, but not other cells, or (ii) substrate specificity, where VDJ genes mutate, but not other genes. Exonuclease activity will assayed by primer extension to map the sites where nicks have occurred. Exonuclease activity will be assayed by degradation of radiolabeled DNA. Proteins will be purified and tested in vitro to study the mechanism of mutation.