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[ subject:"Immunology." ]
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Recruitment of RAG1 and RAG2 to Chro...
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Shetty, Keerthi.
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Recruitment of RAG1 and RAG2 to Chromatinized DNA During V(D)J Recombination.
紀錄類型:
書目-電子資源 : Monograph/item
正題名/作者:
Recruitment of RAG1 and RAG2 to Chromatinized DNA During V(D)J Recombination./
作者:
Shetty, Keerthi.
面頁冊數:
132 p.
附註:
Source: Dissertation Abstracts International, Volume: 76-11(E), Section: B.
Contained By:
Dissertation Abstracts International76-11B(E).
標題:
Immunology. -
電子資源:
http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3663663
ISBN:
9781321963113
Recruitment of RAG1 and RAG2 to Chromatinized DNA During V(D)J Recombination.
Shetty, Keerthi.
Recruitment of RAG1 and RAG2 to Chromatinized DNA During V(D)J Recombination.
- 132 p.
Source: Dissertation Abstracts International, Volume: 76-11(E), Section: B.
Thesis (Ph.D.)--Yale University, 2015.
This item is not available from ProQuest Dissertations & Theses.
V(D)J recombination is essential for the generation of mature B and T lymphocytes. The process is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that flank the antigen receptor gene segments and consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Previous work from our lab revealed parameters of RAG binding at steady-state levels in primary mouse lymphocytes but did not capture the dynamics of the RAG-chromatin interactions. Here, I use RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for efficient RAG recruitment to chromatin.
ISBN: 9781321963113Subjects--Topical Terms:
611031
Immunology.
Recruitment of RAG1 and RAG2 to Chromatinized DNA During V(D)J Recombination.
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V(D)J recombination is essential for the generation of mature B and T lymphocytes. The process is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that flank the antigen receptor gene segments and consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Previous work from our lab revealed parameters of RAG binding at steady-state levels in primary mouse lymphocytes but did not capture the dynamics of the RAG-chromatin interactions. Here, I use RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for efficient RAG recruitment to chromatin.
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Using a catalytic mutant form of RAG1 to prevent DNA cleavage and recombination, I did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Pc genes over a 48 hour time course. Using retroviral recombination substrates, I found that RAG1 was recruited least well to substrates lacking an RSS or containing a single RSS, better to substrates with two 12RSSs or two 23RSSs, and most efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding and a weak contribution by the heptamer. Correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly recreates the nonamer, was ineffective in recruiting RAG1. My findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage and recombination but also the efficiency of RAG recruitment to chromatin.
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Determining the number of RAG molecules per thymocyte may also help us better understand the regulation of RAG binding with one another and with DNA. Previous estimates of nuclear RAG1 and RAG2 concentrations in developing lymphocytes were based on crude experimental procedures and assumptions. In an attempt to obtain more accurate numbers, I performed quantitative western blotting of whole cell extracts prepared from mouse thymus. The RAG1 and RAG2 signal intensities of these extracts were compared to those of purified RAG protein standards with experimentally determined concentrations. The results revealed an average value for RAG1 of 1,800 molecules (monomer) per cell, and for RAG2, 15,300 molecules per cell. These values provide insight into the regulation of RAG1RAG2 interactions as well as RAG-DNA binding and cleavage during V(D)J recombination.
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