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Originally published In Press as doi:10.1074/jbc.M007375200 on January 18, 2001

J. Biol. Chem., Vol. 276, Issue 15, 11838-11843, April 13, 2001
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Identification and Characterization of an Entamoeba histolytica Upstream Regulatory Element 3 Sequence-specific DNA-binding Protein Containing EF-hand Motifs*

Carol A. GilchristDagger , Chris F. Holm§, Molly A. HughesDagger , Joanna M. Schaenman§, Barbara J. MannDagger §, and William A. Petri Jr.Dagger §||

From the Departments of Dagger  Internal Medicine, § Microbiology, and  Pathology, University of Virginia, Charlottesville, Virginia 22908

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca2+-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in the E. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

* This work was supported by National Institutes of Health Grant AI 37941.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF291721.

|| A Burroughs Wellcome Fund Scholar in Molecular Parasitology. To whom correspondence should be addressed: W. A. Petri, Jr., Univeristy of Virginia Health System, MR4 Bldg., Rm. 2115, P.O. Box 801340, Charlottesville, VA 22908-1340. Tel.: 804-924-5621; Fax: 804-924-0075; E-mail: wap3g@virginia.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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