Genes encoding the two critical types of antigen recognition molecules in the immune system immunoglobulin (Ig) and T cell receptor (TCR), are assembled during lymphocyte development by a novel, highly regulated site-specific DNA recombination reaction known as V(D)J recombination. The combinatorial joining of gene segments allows the immune system to encode near limitless antigen recognition capability with only a modest genetic investment. The developing lymphocyte is faced with the problem, however, of regulating this reaction to produce functional Ig or TCR while avoiding the consequences of the genomic instability inherent in somatic recombination. Several new techniques have been developed to study the details of the V(D)J recombination reaction pathway and the molecular mechanisms which regulate this reaction. The investigator has devised a series of PCR-based assays which allow detection of V(D)J recombination reaction intermediates consisting of specifically broken DNA molecules. These assays will be used to determine the precise structure of broken DNA coding ends, their location within nuclei of cells undergoing gene rearrangement, the accessibility of these ends, and the identities of proteins which are bound to them. The hypothesis that aberrant V(D)J recombination is involved in the etiology of leukemia associated chromosomal translocation will be tested by looking for DNA breaks at cryptic recombination signal sequences (RSSs) in proto-oncogenes. Recently, researchers at the NIH reported the first in vitro system capable of specific recognition and cleavage of rearranging genes. This system will be used to address the question of how targeting of the recombinase is regulated by performing in vitro cleavage assays on nuclei isolated from various lymphoid precursors. The idea that chromatin structure dictates the choice of target loci by the recombinase will also be tested. Finally, to determine whether the minimally active """"""""core"""""""" domains of recombinase genes RAG-1 and RAG-2 can rescue B-cell development in either RAG-I or RAG-2 deficient transgenic mice, mice expressing these proteins will be made. Lastly, the investigator will screen for proteins in lymphoid precursors which interact with the unessential domains of the RAGs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI040227-02
Application #
2442713
Study Section
Special Emphasis Panel (ZRG2-ALY (01))
Project Start
1996-07-01
Project End
2000-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Degner, Stephanie C; Verma-Gaur, Jiyoti; Wong, Timothy P et al. (2011) CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells. Proc Natl Acad Sci U S A 108:9566-71
Guo, Chunguang; Yoon, Hye Suk; Franklin, Andrew et al. (2011) CTCF-binding elements mediate control of V(D)J recombination. Nature 477:424-30
Sukumar, Selvakumar; Schlissel, Mark S (2011) Receptor editing as a mechanism of B cell tolerance. J Immunol 186:1301-2
Vettermann, Christian; Schlissel, Mark S (2010) Allelic exclusion of immunoglobulin genes: models and mechanisms. Immunol Rev 237:22-42
Curry, John D; Schlissel, Mark S (2008) RAG2's non-core domain contributes to the ordered regulation of V(D)J recombination. Nucleic Acids Res 36:5750-62
Hewitt, Susannah L; Farmer, Deborah; Marszalek, Katarzyna et al. (2008) Association between the Igk and Igh immunoglobulin loci mediated by the 3'Igk enhancer induces 'decontraction'of the Igh locus in pre-B cells. Nat Immunol 9:396-404
Bates, Jamie Geier; Cado, Dragana; Nolla, Hector et al. (2007) Chromosomal position of a VH gene segment determines its activation and inactivation as a substrate for V(D)J recombination. J Exp Med 204:3247-56
Curry, John D; Schulz, Danae; Guidos, Cynthia J et al. (2007) Chromosomal reinsertion of broken RSS ends during T cell development. J Exp Med 204:2293-303
Curry, John D; Geier, Jamie K; Schlissel, Mark S (2005) Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis. Nat Immunol 6:1272-9

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