Analysis of immunoglobulin S gamma 3 recombination breakpoints by PCR: implications for the mechanism of isotype switching

The molecular mechanism of immunoglobulin switch recombination is poorly understood, Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes, previously we showed that switch recombination breakpoints cluster to it defined subregion in the S gamma 3, S gamma 1 and S gamma 2b tandem repeats. We have developed a strategy for direct amplification of S mu/S gamma 3 composite fragments as well as S mu and S gamma 3 regions by PCR. This assay has been used to analyze the organization of S mu, S gamma 3 and a series of S mu/S gamma 3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells, DNA sequence analysis of the switch fragments showed direct joining of S mu and S gamma 3 without deletions or duplications, Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the S gamma 3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction.