THE NADP-DEPENDENT GLUTAMATE-DEHYDROGENASE OF GIARDIA-LAMBLIA - A STUDY OF FUNCTION, GENE STRUCTURE AND EXPRESSION

Giradia lamblia is an interesting organism in several respects. Giardi a is a medically important protozoan parasite and its location in the phylogenetic tree is at a critical and informative position. Character ization of its subcellular structure and rRNA sequences suggest that g iardia is one the most primitive eukaryotes known, Giardia has an anae robic metabolism that uses NADP-glutamate dehydrogenase (GDH) along wi th alanine aminotransferase to dissipate excess reducing power and to maintain a intracellular balance of NAD(P)-NAD(P)H through the convers ion of pyruvate to alanine. The giardia NADP-GDH is encoded by a singl e copy gene in the haploid genome. Transcript mapping and comparisons of the cDNA and genomic clones for the GDH gene have indicated the abs ence of introns, RNA editing, and transsplicing. Transcripts from the GDH gene, as in other giardia genes, have short untranslated sequences and are initiated only three to six nucleotides in front of the ATG t ranslation initiation codon. Promoter elements normally associated wit h RNA polymerase II transcription in higher eukaryotes are not found i n the upstream sequence of the GDH gene or in other giardia genes. How ever, two novel sequence motifs were identified in these upstream sequ ences; one is the AT-rich element and the other is the CAAAT element. Their conservation and location relative to the sites of transcription initiation suggest that these elements may be involved in the regulat ion of transcription. A 68 kD protein, called POT, binds to the TTT tr inucleotides found on either one or both strands of the DNA encoding t hese elements from the upstream region of the GDH gene. We suggest tha t POT helps to denature the DNA at the promoter region and to particip ate in the assembly of the RNA polymerase II pre-initiation complex.