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). Full-length cDNA and genomic clones for GS have been isolated. Fusion constructs have been made between the GS promoter region and a reporter gene, chloramphenicol acetyltransferase (CAT). Sequence analysis has identified consensus sequences for the binding sites of AP2, SP1, GRE, and a silencer. Transfection of the fusion constructs into Primary astrocytes and a variety of cell lines has demonstrated the functional importance of these regions. Site-directed mutagenesis of these sites has confirmed the identity of the sites, and has shown interactions between the sites. Astrocytic message level for GS are increased by coculture with glutamatergic cerebellar granule cells. This effect can also be produced by culturing the astrocytes with either fixed granule cells or membranes prepared from them. Transfectional studies with promoter fusion constructs will demonstrate the cis-acting elements responsible for this phenomenon. Pulse-chase experiments are being used to determine the potential role of message stabilization. A 4 kbp partial cDNA clone for GA, and genomic clones for these sequences have also been isolated. Gene-specific cDNA libraries for GA have been made in an attempt to clone the 5' end of the GA message, and Northern and 51 protection assays used to examine the tissue distribution and developmental expression of GA. GA is found at highest levels in brain and kidney, and is absent from liver. In the brain GA message appears near the time of birth and reaches a transient peak at two weeks postnatally, the time at which cerebellar granule cells are making synaptic contact
