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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
Identification and Characterization of an
Entamoeba histolytica Upstream Regulatory Element 3 Sequence-specific DNA-binding Protein Containing EF-hand Motifs*
Carol A.
Gilchrist ,
Chris F.
Holm§,
Molly A.
Hughes ,
Joanna
M.
Schaenman§,
Barbara J.
Mann §, and
William A.
Petri Jr. §¶
From the Departments of 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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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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