I propose to continue studying how gene expression is regulated during the differentiation of eukaryotic cells using Dictyostelium discoideum as an experimental system. I propose to identify specific colonies containing the structural genes for two enzymes of Dictyostelium, from a library of colonies of E. coli containing fragments of the Dictyostelium DNA. These enzymes, uridine diphosphoglucose pyrophosphorylase (UDPGP) and alkaline phosphatase accumulate at specific developmental stages. They and most proteins represent only a small fraction of the cellular protein. Nevertheless, at least UDPGP is essential for normal development. In order to study the expression of genes whose enzyme proteins accumulate at low levels, we have developed a novel high resolution, denaturing slab gel technique. It allows renaturation and localization of a number of enzymes (including UDPGP) in situ, from crude extracts. With this technique it is possible to assay the mRNA of UDPGP and to measure its accumulation at different developmental stages. In addition, it will be valuable for screening the bank for colonies which contain the gene for UDPGP. This procedure has also enabled us to discover, in crude extracts, an isozyme of UDPGP which appears late in development. I propose to investigate its molecular origin and its distribution in the two major tissues of this organism. We are also characterizing the enzyme UDPGP from a number of mutants with reduce activity, to aid in distinguishing structural gene mutants from those that may be regulatory. This molecular genetic approach should elucidate some of the mechanisms which are involved in the differentiation of eukaryotic cells.