A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
| Autor: | Karen Kage, Emily Klotz, Terence E. Martin, Grazyna Bozek, John Hackett, Ursula Storb |
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| Rok vydání: | 1998 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Immunology Immunoglobulin Variable Region Somatic hypermutation Mice Transgenic Biology medicine.disease_cause Immunoglobulin kappa-Chains Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine RNA polymerase medicine Animals Immunology and Allergy Transgenes Ig genes Deoxyribonucleases Type II Site-Specific Gene 030304 developmental biology Genetics 0303 health sciences Mutation Binding Sites Base Sequence RNA Articles RNA secondary structure RNA polymerase pausing Molecular biology somatic hypermutation hot spots Mutagenesis Insertional Restriction site Restriction enzyme chemistry Immunoglobulin Joining Region Nucleic Acid Conformation DNA 030215 immunology |
| Zdroj: | The Journal of Experimental Medicine |
| ISSN: | 1540-9538 0022-1007 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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