A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript.
Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA. stor@midway.uchicago.edu
U
U
Storb
E L
EL
Klotz
J
J
Hackett
K
K
Kage
G
G
Bozek
T E
TE
Martin
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D019076:Transgenes
D018076:DNA, Complementary; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D012313:RNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases