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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 ini...

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Autores principales: Storb, Ursula, Klotz, Emily L., Hackett, John, Kage, Karen, Bozek, Grazyna, Martin, Terence E.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1998
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213363/
https://www.ncbi.nlm.nih.gov/pubmed/9705951
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author Storb, Ursula
Klotz, Emily L.
Hackett, John
Kage, Karen
Bozek, Grazyna
Martin, Terence E.
author_facet Storb, Ursula
Klotz, Emily L.
Hackett, John
Kage, Karen
Bozek, Grazyna
Martin, Terence E.
author_sort Storb, Ursula
collection PubMed
description 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.
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spelling pubmed-22133632008-04-16 A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript Storb, Ursula Klotz, Emily L. Hackett, John Kage, Karen Bozek, Grazyna Martin, Terence E. J Exp Med Articles 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. The Rockefeller University Press 1998-08-17 /pmc/articles/PMC2213363/ /pubmed/9705951 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Storb, Ursula
Klotz, Emily L.
Hackett, John
Kage, Karen
Bozek, Grazyna
Martin, Terence E.
A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title_full A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title_fullStr A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title_full_unstemmed A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title_short A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
title_sort hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the rna transcript
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213363/
https://www.ncbi.nlm.nih.gov/pubmed/9705951
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