The Journal of Experimental Medicine
Torrey Pines Biolabs
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 231K)
Right arrow PPT slides of all figures
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new content in the JEM
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Storb, U.
Right arrow Articles by Martin, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Storb, U.
Right arrow Articles by Martin, T. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?
© The Rockefeller University Press, 0022-1007/1998/8/689/ $5.00
The Journal of Experimental Medicine, Volume 188, Number 4, August 17, 1998 689-698

Articles

A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript

Ursula Storb*, Emily L. Klotz{ddagger}, John Hackett, Jr.*, Karen Kage*, Grazyna Bozek*, and Terence E. Martin*

From the * Department of Molecular Genetics and Cell Biology and the {ddagger} Committee on Immunology, University of Chicago, Chicago, Illinois 60637

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.

Key Words: Ig genes • somatic hypermutation • RNA secondary structure • hot spots • RNA polymerase pausing

Address correspondence to Ursula Storb, Department of Molecular Genetics and Cell Biology, University of Chicago, 920 E 58th St., Chicago, IL 60637. Phone: 773-702-4440; Fax: 773-702-3172; E-mail: stor{at}midway.uchicago.edu

We are greatly indebted to Phil Schumm for the statistical analysis. We are grateful to Peter Engler, Nancy Michael, Larry Loeb, David Roth, and Kevin Struhl for critical reading of the manuscript.

Dr. Klotz's current address is NCI, NIH, Bethesda, MD 20892. Dr. Hackett's current address is Abbott Laboratories, North Chicago, IL 60064.

Abbreviations used: C, constant; D, diversity; EPS, alternating EcoRV and PvuII restriction enzyme sites; J, joining; MuF, mutator factor; pol, RNA, polymerase II; V, variable.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?




  Home | Help | Feedback | Subscriptions | Archive | Search
TABLE OF CONTENTS