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Understanding the Immunoglobulin Locus Specificity of Hypermutation

  • Vera Batrak
  • Artem Blagodatski
  • Jean-Marie BuersteddeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contribute to the primary Ig diversity repertoire formation in some species. How the hypermutation activity is specifically targeted to the Ig loci is a long-standing debate. Here we describe a new experimental approach to investigate the locus specificity of Ig hypermutation using the chicken B-cell line DT40. One feature is the use of a green fluorescent protein (GFP) gene as a mutation reporter. Some nucleotide changes produced by somatic hypermutation can cripple the GFP gene which leads to a decrease or loss of the green fluorescence. Therefore such changes can be easily quantified by fluorescence-activated cell sorting (FACS). Another advantage of this approach is the targeted integration of the mutation reporter into a defined chromosomal position. This system allowed us to identify a 10 kb sequence within the Ig light chain (IgL) locus, which is both necessary and sufficient to activate hypermutation in the neighboring reporter gene. We have called this sequence Diversification Activator (DIVAC) and postulated that similar cis-acting sequences exist in the heavy and light chain Ig loci of all jawed vertebrate species. Our experimental system promises further insight into the molecular mechanism of Ig hypermutation. For example, it may be possible to identify smaller functional motifs within DIVAC and address the role of putative transacting binding factors by gene knock-outs.

Key words

Somatic hypermutation immunoglobulin gene AID B cell DT40 DIVAC 

Notes

Acknowledgments

AB was supported by the grant no. 02.740.11.5016 from the Russian Ministry of Science.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Vera Batrak
    • 1
  • Artem Blagodatski
    • 2
  • Jean-Marie Buerstedde
    • 3
    Email author
  1. 1.HildesheimGermany
  2. 2.Institute of Protein Research, Russian Academy of Sciences, Russian FederationMoscowRussia
  3. 3.Istra, Moscow RegionRussia

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