In the cerebellum of the mouse the expression of the structural gene (Gdc-1) for the enzyme glycerol-3-phosphate dehydrogenase (GPDH) is modulated by unlinked genes. The most interesting regulatory gene, Gdcr-1, acts by increasing the level of Gdc-1 transcripts. This regulatory gene has two interesting properties. First, it seems to act only in the cerebellum and second, alleles at Gdcr-1 act preferentially on alleles at Gdc-1, i.e., the Gdcr-1c allele preferentially enhances expression of the Gdc-1c allele. In this application we seek to identify sites within or flanking the Gdc-1 gene which is involved in determining the cerebellar specific expression. Available for this project are Gdc-1 genomic clones which were isolated and sequenced in the previous grant period. The experimental approach is to first identify DNA sequences within the Gdc-1 gene or its flanking region which enhance transcription in a transient expression assay. These regions will be evaluated further as the cerebellar regulatory region on the basis of genetic variability and the cerebellar specific expressions in the transient expression assays. A second set of experiments aims to map genetic location of the Gdcr-1 gene and to evaluate whether Gdcr-1 acts in reaggregating cultures of mouse cerebellar cells. A third set of experiments seeks to clone the cDNA and the gene for the embryonic GPDH isozyme which is located on Chromosome 9. The experimental approach is to make a lambdagt 11 cDNA library from rabbit heart and screen for a protein in this library which cross-reacts with anti-mouse embryonic GPDH. The rationale for the experiment is that rabbit heart has isozymes of GPDH which have properties similar to the mouse embryonic enzyme. Once the Gdc-2 gene has been cloned, we will have a chance to identify controlling regions in and around the gene which are involved in expression. A comparison of the controlling regions of Gdc-2 with those associated with Gdc-1 may provide insight into the structural requirement for genes expressed in the embryo as compared to those expressed in the adult.
