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Nucleic Acids Research 2004 32(14):4313-4321; doi:10.1093/nar/gkh771
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Published online 10 August 2004

Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved

Transcriptional control of RAD51 expression in the ciliate Tetrahymena thermophila

Joshua J. Smith, Eric S. Cole1 and Daniel P. Romero*

Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA and 1 Department of Biology, St Olaf College, Northfield, MN 55057, USA

* To whom correspondence should be addressed. Tel: +1 612 624 8997; Fax: +1 612 625 8408; Email: romero{at}med.umn.edu
Present address: Joshua J. Smith, University of Virginia Health Systems, Biochemistry and Molecular Genetics, Charlottesville, VA 22908, USA

Received June 28, 2004; Revised and Accepted July 27, 2004

The expression of Rad51p, a DNA repair protein that mediates homologous recombination, is induced by DNA damage and during both meiosis and exconjugant development in the ciliate Tetrahymena thermophila. To completely investigate the transcriptional regulation of Tetrahymena RAD51 expression, reporter genes consisting of the RAD51 5' non-translated sequence (5' NTS) positioned upstream of either the firefly luciferase or green fluorescent protein coding sequences have been targeted for recombination at the macronuclear btu1-1 (K350M) locus of T. thermophila strain CU522. Expression from RAD51-luciferase reporter constructs has been directly quantified from transformant whole cell lysates. Luciferase is induced to maximum levels in transformants harboring the full-length RAD51-luciferase reporter gene following exposure to DNA damaging UV irradiation. A series of truncations, deletions, insertions, substitutions and inversions of the RAD51 5' NTS have led to the identification of three distinct transcriptional promoter elements. The first of these sequence elements is required for basal levels of transcription. The second modulates expression in the absence of DNA damage, whereas the third ensures increased RAD51 transcription in response to DNA damage and during meiosis. Tetrahymena RAD51 is tightly regulated through these transcriptional elements to produce the appropriate expression during conjugation, and in response to DNA damage.


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