The Ig heavy chain switch region is a hotspot for insertion of transfected DNA.
The Ig heavy chain class switch usually occurs by breaking and rejoining DNA in the switch (S) regions, which consist of tandemly repeated sequences 5' of the constant region exons. Various studies have suggested that S DNA can also recombine with non-S sequences. To measure the frequency of such recombination events, the hybridoma cell line igm692, a deletion mutant that lacks the C mu 1 and C mu 2 exons and the 3' end of the S mu region, was transfected with a fragment bearing the C mu 1-2 exons, but no S mu DNA. Insertion of this fragment into the residual VDJ-C mu intron of igm692 can restore a functional mu gene, yielding a transformant that is detected as a plaque-forming cell (PFC). PFC comprise approximately 8 x 10(-7) of the surviving transfected cells. In 10 of 12 PFCs, the C mu 1-2 fragment inserted into the 2.5-kb residual S mu region, whereas insertion in two cases occurred in the 3.5-kb segment 5' of S mu. Using a PCR assay to measure the frequency of insertion of the transferred fragment elsewhere in the hybridoma genome, we found that approximately 9% of the surviving transfected cells had stably acquired the C mu 1-2 fragment. These results indicate that the S mu region is approximately 100-fold more recombinogenic than the average genomic site, and approximately 7-fold more recombinogenic than the non-S mu segment of the residual VDJ-C mu, i.e., the S mu region is a hotspot for insertion of transfected DNA.
Baar
J
J
Department of Immunology, University of Toronto, Ontario, Canada.
Shulman
M J
MJ
D001483:Base Sequence; D002460:Cell Line; D017578:Immunoglobulin Class Switching; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D011995:Recombination, Genetic; D014162:Transfection
D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007075:Immunoglobulin M; D012333:RNA, Messenger