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The NADP-dependent glutamate dehydrogenase of Giardia lamblia: a study of function, gene structure, and expression Yee, Janet
Giardia lamblia is an interesting organism in several respects. Not only is it a medically important protozoan parasite, but its location in the phylogenetic tree is at a critical and informative position. Characterization of its subcellular structure and rRNA sequences suggest that Giardia is one the most primitive eukaryotes known. Giardia has an anaerobic metabolism that uses a NADP-dependent glutamatede hydrogenase (GDH) along with alanine aminotransferase to maintain an intracellular balance of NAD(P)+-NAD(P)H through the conversion of pyruvate to alanine. In the initial part of this study, the Giardia NADP-GDH gene was cloned and characterized. The Giardia NADP-GDH is encoded by a single copy gene in the haploid genome. Transcript mapping and comparisons of the cDNA and genomic clones for the GDH gene did not detect the presence of introns or transsplicing. GDH transcripts have short untranslated sequences and are initiated only three to six nucleotides in front of the ATG translation initiation codon. In the latter part of this study, the 5' flanking sequence of the Giardia GDH gene was analyzed to identify possible promoter elements since core promoter elements normally associated with RNA polymerase II transcription in higher eukaryotes are not found in the upstream sequence of the GDH gene or in other Giardia genes. Two novel sequence motifs were identified, an AT-rich element and a Giardia CAAT-box-like sequence called the g-CAB element. Their conservation and locations relative to the sites of transcription initiation suggest that these elements may be involved in the regulation of transcription. A 68 kD protein binds to the TTT trinucleotides found on either one or both strands of the DNA encoding these elements from the upstream region of the GDH gene. This 68 kD protein is referred to as POT for its ply(T) binding ability. The function of POT may be to help denature the DNA at the promoter region and to participate in the assembly of the RNA polymerase II pre-initiation complex in Giardia.
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