An understanding of the genetic mechanisms controlling differentiation will require that specific genes can be analyzed at the levels of transcription and translation. As a model system for neural differentiation we are analyzing the isozymes of L-glycerol 3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) in the developing cerebellum of mice under in vivo and in vitro conditions. Several requirements for such an analysis have been established, these include the availability of antibodies to the individual isozymes, the identification of tissues in which the adult isozyme represents up to 0.8 percent of the total protein, and the ability to measure enzyme synthesis in vitro. In addition, genetic variants of this isozyme that effect the protein structure and enzyme protein levels are available and the transition of isozyme expression during development can be shown to occur in primary cell cultures and established cell lines. The specific questions we wish to ask are: (1) Is the variation in adult isozyme expression, during development, among different genotypes, and in diseased states controlled by changes in the amount of specific mRNA? (2) At what point of transcription or translation is the control of mRNA levels exerted? (3) Are the adult and embryonic isozymes of GPHD coded for by separate structual genes? Our approach to this problem will be to measure both translatable and hybridizable mRNA. The analysis of hybridizable mRNA and the isolation of radioactive nuclear RNA for measurements of transcriptional rates will require a cDNA for GPHD. Our plan is to purify the mRNA for GPHD until it is the major species in the population, clone the cDNA molecules of this population, and then detect clones for GPHD cDNA by in situ hybridization. In addition to the above experiments radoactive GPHD cDNA will be used to detect clones for the GPHD gene in a genomic mouse library. Since the isozymes of GPHD appear to be a very similar structurally we plan to use the adult GPHD cDNA to find the gene for the embryonic isozyme.
