A study of immunoglobulin heavy chain 3' enhancer function within chromatin.
Item
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Title
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A study of immunoglobulin heavy chain 3' enhancer function within chromatin.
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Identifier
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AAI9986378
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identifier
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9986378
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Creator
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Shi, Xuerong.
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Contributor
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Adviser: Laurel Eckhardt
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Date
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2000
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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The immunoglobulin heavy chain (IgH) locus is controlled by multiple regulatory elements including IgH promoter and intronic enhancer (Emu) located within the IgH transcription unit, and 3'IgH enhancers (Hs3a, Hs1,2, Hs3b, and Hs4) residing downstream of IgH coding sequences. Emu plays a central role in VDJ recombination and activates IgH gene transcription at the earlier B cell stages. The 3' IgH enhancers are believed activate/maintain IgH gene transcription at the later B cell stages and may also play a role on heavy chain class switch recombination.;To assess directly the regulatory activity of the first identified 3 'IgH enhancer, Hs1,2, we deleted this enhancer from the IgH locus of an Ig-secreting cell line that already lacked Emu. We found that Hs1,2 was essential for IgH gene transcription in this cell line.;To study further the regulatory activity of this and the other 3 'IgH enhancers in the context of chromatin, we created IgH mini-loci (Iggamma2b transgenes under control of the 3'IgH enhancers) to manipulate these 3' enhancers. The mini-loci were used to stably transform both a surface-Ig positive cell line and an Ig-secreting cell line. After stable expression of the loci had been achieved in the respective cell lines, we induced enhancer deletions within the mini-loci. We found that the hs3a/hs1,2 and the hs3b/hs4 pairs were functionally redundant in the surface Ig+ cell line but that the hs3b/hs4 pair played a dominant role in maintaining reporter gene expression in the Ig-secreting cell line. Our results provide evidence to support the importance of the 3' IgH enhancers in IgH transcription at the later B cell stages, and support and extend emerging models of the IgH locus in which complex and shifting modes of control regulate this locus over the course of B lymphocyte development and antigen-stimulated differentiation.;Finally, we exploited the possibility that the 3'IgH enhancers are able to interact with one another and/or with IgH promoters through an association with the nuclear matrix. We assayed most of a 34 kb region encompassing the 3'IgH enhancers for the presence of matrix attachment regions (MARs). While our assays confirmed the presence of MARs adjacent to the intronic enhancer, Emu, we saw no evidence of MARs in the 3'IgH enhancer region.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
