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J Biol Chem, Vol. 275, Issue 15, 11432-11439, April 14, 2000
From the Laboratory of Parasitic Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892 and the § Department of
Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, Canada
We studied gene expression in the ancient
eukaryote, Giardia lamblia, by taking advantage of assays
developed recently in our laboratory, which allow new genetic analyses
of this organism. We examined the transcription of a 2.2-kilobase
segment of the Giardia genome that contains the
glutamate dehydrogenase (GDH) gene and a
portion of a second open reading frame encoding an uncharacterized
gene. Nuclear run-on analyses showed that the genes are transcribed as
two separate units spaced less than 200 base pairs apart, and
transcription of the GDH gene initiates just 3-6
nucleotides upstream of its translation start codon. We characterized
the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive
the expression of a luciferase reporter gene. By deletion and
mutational analyses, we localized promoter function to three motifs
within a 50-base pair region of the GDH upstream sequence.
Using band shift assays and UV cross-linking, we demonstrated specific
binding of a 68-kDa protein from Giardia nuclear extracts
to short poly(T) tracts contained within two of the sequence motifs on
single-stranded DNA from the promoter region. This report describes one
of the first functional gene promoter and its cognate DNA-binding
protein in this primitive eukaryote.
Transcriptional Analysis of the Glutamate Dehydrogenase Gene in
the Primitive Eukaryote, Giardia lamblia
IDENTIFICATION OF A PRIMORDIAL GENE PROMOTER*
,
*
A portion of this work was supported by Grant MT6340 from
the Medical Research Council of Canada (to P. P. D.).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.
Present address: Dept. of Biology, Trent University, 1600 West
Bank Dr., Peterborough, Ontario K9J 7B8, Canada. To whom correspondence should be addressed: Tel.: 705-748-1048; Fax: 705-748-1625; E-mail: jyee@trentu.ca.
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