The goal of this project is to study the structure, function, and regulation of the genes for the astrocytic enzyme glutamine synthetase (GS), and the neuronal enzyme phosphate-activated glutaminase (GA). Fusion constructs have been made between the GS promoter and the chloramphenicol acetyltransferase (CAT) reporter gene. Four regions of regulatory importance have been characterized in the GS promoter: a modulator region with homology to AP2, a GRE region, a silencer region, and a second region which inhibits transcription. Polymerase chain reaction (PCR)-based site-directed mutagenesis of these sites has demonstrated their importance in GS regulation, and the interactions between them. The modulator site is required for activity of both the GRE and silencer sites. The occult inhibitory region appears to act independently. Double-stranded oligonucleotide probes for each of these sites have been used in electrophoretic mobility shift assays (EMSA). Proteins from nuclear extracts of positively responding primary astrocytes and HepG2 hepatoma cells can bind all three sites, while those from a negatively responding cell line, HeLa, can only bind at the GRE site. The modulator site does not bind purified AP2 protein, although it contains partial sequence homology to it. The GRE site binds the DNA-- binding fragment from the glucocorticoid receptor, and can compete for binding to an idealized GRE site. Biotinylated oligonucleotide probes specific for each site have been coupled to magnetic beads for affinity purification of the trans-acting factors binding to these sites. Incubation of these probes with nuclear extract from HepG2 cells has partially depleted the extracts of the silencer element, as determined by EMSA. Cloning the GA gene has resulted in extension of the primary clones through the rapid amplification of cohesive ends (RACE) procedure. Screening a rat hippocampal library with this probe resulted in isolation of a cDNA clone for GA which extends well into the 5' untranslated region. Reverse transcription followed by PCR (RT-PCR) has been used to determine the developmental and tissue-specific expression of the GA gene.
