We propose to investigate the molecular mechanisms of isotype switch recombination in the immunoglobulin heavy chain locus of the mouse. Switch recombination normally occurs during an immune response to change the effector function of an antibody molecule while retaining its specificity for antigen recognition. In vitro, a switch from expression of IgM to IgG3 or IgG1 can be specifically and efficiently induced in a primary lymphocyte culture by treatment with the nonspecific mitogen, lipopolysaccharide (LPS), or LPS in combination with the lymphokine interleukin 4 (IL4). Isotype switching is mediated by a recombination event between internally repetitious DNA sequences, called switch regions, which lie upstream from each constant region and presumably play a role in directing inducible as well as constitutive components of the cellular recombination machinery to effect a recombination event. We plan (1) to sequence a number of Smu/Sgammal recombination joints in primary lymphocytes specifically induced to switch with LPS + IL4; (2) to identify proteins in nuclear extracts of induced lymphocytes that can bind to either the Smu or Sgammal switch regions, using a sensitive gel mobility shift assay; (3) to localize the binding sites of the proteins by DNA footprinting; (4) to use a biochemical assay to test the nuclear extracts for switch region- specific endonuclease and/or topoisomerase activities; and finally (5) to achieve switch recombintion in extracts from induced primary lymphocytes, using a specially designed test construct and a very sensitive biological assay, and to use this in vitro system to define the proteins and DNA sequences essential for isotype switch recombination. We hope that these experiments will lay the groundwork for a detailed molecular understanding of site specific recombination in mammalian systems.
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| Yabuki, Munehisa; Ordinario, Ellen C; Cummings, W Jason et al. (2009) E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification. J Immunol 182:408-15 |
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| Duquette, Michelle L; Pham, Phuong; Goodman, Myron F et al. (2005) AID binds to transcription-induced structures in c-MYC that map to regions associated with translocation and hypermutation. Oncogene 24:5791-8 |
| Maizels, Nancy (2005) Immunoglobulin gene diversification. Annu Rev Genet 39:23-46 |
| Larson, Erik D; Duquette, Michelle L; Cummings, W Jason et al. (2005) MutSalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions. Curr Biol 15:470-4 |
| Larson, Erik D; Cummings, W Jason; Bednarski, David W et al. (2005) MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification. Mol Cell 20:367-75 |
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