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Characterization of novel DNA binding activities at the immunoglobulin 3' enhancer

Author

Matthew Hoyt Milnes, University of the Pacific

Date of Award

2000

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Craig Vierra

First Committee Member

Gregg Jongeward

Second Committee Member

Paul Richmond

Abstract

Transcription of the immunoglobulin heavy chain is influenced, in part, by an intronic enhancer region located 3' to the V,D, and J gene segment regions. The regulatory elements within this enhancer show high levels of sequence homology between mouse, rat, rabbit, and human, indicating their evolutionary significance. SRY, SpiB, LM02, and Rzra have been shown elsewhere to bind recognition sequences found within the evolutionarily conserved regulatory elements of this enhancer. We seek to demonstrate previously unreported individual and cooperative DNA binding activities for I these factors at regulatory' elements within the immunoglobulin 3' enhancer region. To facilitate this investigation, clones representing E2A, p65, SRY, SpiB LM02, ABF-1, and Rzra were retrieved from a plasma cell eDNA library. E2A, p65, and ABF-1 have been previously reported to bind elements within this enhancer. EMSA studies of these factors individually and in concert support previous characterization of DNA binding activities at this enhancer, and demonstrate the hereto unreported individual binding activities of Rzra, LM02, SpiB and SRY. Mixing studies with LM02 and SpiB show that when simultaneously present, these factors block the binding activity of one another. Furthermore, SpiB and LM02 are capable of blocking the in vitro DNA binding activity ofE2A and p65, presumably through the formation of a non-DNA binding complex. Mixing studies with Rzra demonstrate it to be a high affinity DNA binding factor capable of blocking p65 and E2A binding activity without relinquishing its own activity. We suspect this blocking activity to be a result of steric hinderance based on the close linear proximity of these factors recognition sequences.

Pages

78

Recommended Citation

Milnes, Matthew Hoyt. (2000). Characterization of novel DNA binding activities at the immunoglobulin 3' enhancer. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/535