Expression and recombinase activity of RAG 1 and two splice variants of RAG 2 in mature human primary tonsilar B lymphocytes.

摘要

RAG 1 and RAG 2 are required for the rearrangement of both the immunoglobulin (Ig) and T cell receptor genes. Their expression was first thought only to occur during the immature stages of B and T cell development. Studies in our lab using a human mature B cell line OCI LY8 and a unique set of its variants demonstrated variation in expression of both the BCR and the RAG 1 and RAG 2 genes. Evidence employing mouse models demonstrated an unexpected re-expression of the RAG genes in mature mouse B cells activated in vitro and in peripheral lymph node germinal centres of immunized mice, indicating the potential for further Ig gene rearrangements. In this thesis, I describe experiments aimed at establishing whether re-expression of the RAG genes occurs in humans. A Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) assay was developed and optimized to determine expression of the RAG genes in human peripheral lymphoid tissue. I found that RAG 1 transcripts, as well as transcripts of two RAG 2 splice variants were detectable in human tonsilar B lymphocytes and are considered to be responsible for the dsDNA breaks observed in these cells under linker-mediated PCR (LM-PCR) analysis.