DNA REPAIR AND SOMATIC MUTATION IN ANTIBODY VARIABLE GENES

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A correlation between a genetic defect in DNA repair and lack of hypermutation would be very significant, since there are no in vivo models to study mutation. Three knockout lines of mice have been obtained: PMS2 and MSH2 mismatch repair mice, and XPA nucleotide excision repair mice. Homozygous (-/-) knockout and littermate (+/+) mice are being immunized, and DNA will be prepared from B cells. Endogenous Vx and VH genes are being amplified by PCR, cloned, and sequenced. The frequency of mutation in the V and constant genes from knockout and control mice are compared. Preliminary results show different patterns of mutation in normal and mismatch repair deficient mice. Hybridoma cells were irradiated with ultraviolet light. The cells were then grown in vitro and harvested at different time points. DNA was prepared, and incubated with restriction enzymes and T4 endonuclease, which cleaves thymine dimers. Cleaved DNA was separated by gel electrophoresis, and different genes were identified by Southern blot analysis. Current results suggest that V genes have a slower rate of repair than the dihydrofolate reductase gene.