Somatic hypermutation of the variable regions of immunoglobulin genes is responsible for the increase in affinity and changes in fine specificity of antibodies during the secondary response. The detailed mechanisms responsible for this process are largely unknown and until now could only be investigated by examining the sequences of endogenous Ig genes of transgenes in vivo. The applicant has recently established a tissue culture system in which mutation can be studied by reversion analysis, and transfected and manipulated heavy chain genes can undergo V region point mutations at rates as high as 10E-4 to 10E-3/base pair/generation that are comparable to the rates that occur in vivo. He will determine whether the size and borders of the target of mutation in the transfected Ig gene is the same in these cultured cells as it is in vivo. In these cultured cells, mutations occur more frequently in a motif that is a favored target for mutation in vivo. He will use this in vitro system to explore the role of the target sequence, the surrounding sequence environment, and the distance from the promoter in generating these hot spots. Constructs that differ by 1000 fold in their rate of mutation in the NSO cell line will be dissected to identify the cis-acting sequences that are involved in mutation. He will also examine the role of transcription and of the promoter and enhancers in mutation and compare the interactions of the different cis-acting sequences in cell lines that are permissive and non-permissive for mutation. These studies should lead to a better understanding of the mechanisms responsible for the high rate of V region mutation in cultured B cells and, if the characteristics of the process are the same as the normal process, to new insights into the regulation of V region hypermutation in vivo.
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