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Nucleic Acids Research 2005 33(4):1269-1279; doi:10.1093/nar/gki274
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Published online 1 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Molecular characterization of Drosophila NELF

Chwen-Huey Wu, Chanhyo Lee, Ruopeng Fan, Meghan J. Smith, Yuki Yamaguchi1,2, Hiroshi Handa1 and David S. Gilmour*

Department of Biochemistry and Molecular Biology, Center for Gene Regulation, The Pennsylvania State University University Park, PA 16802, USA 1Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Yokohama, Japan 2PRESTO-JST, Tokyo Institute of Technology Yokohama, Japan

*To whom correspondence should be addressed. Tel: +1 814 863 8905; Fax: +1 814 863 7024; Email: dsg11{at}psu.edu

Received January 3, 2005. Revised February 9, 2005. Accepted February 9, 2005.

NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and ß1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.


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