The purpose of the proposed research is the investigation of the regulation of expression of D. melanogaster GTP cyclohydrolase (CH), the enzyme catalyzing the first step in pteridine biosynthesis. These experiments will be part of a long-term study of the regulation of the pteridine biosynthetic pathway with particular emphasis on the control of the developmental specificity of pteridine production. An important feature of the CH system is that a very wide array of phenotypic variation is readily detectable. We have identified several loci that affect the expression of CH. One of these, the Pu locus, appears to be the structural gene for the enzyme. The functions of the other loci are as yet unknown, but they do not behave as structural genes for CH. We have detected several mutations that are tissue or temporally-specific in their effects, some of these in the """"""""non-structural"""""""" loci and some in the Pu locus. Among the proposed experiments are a determination of the precise tissues in which CH activity and protein are found, EMS and Gramma-ray mutagenesis of the Pu region to improve the genetic characterization of the area and to expand the range of phenotypic variants of CH expression at our disposal. We also propose further biochemical analysis of CH with particular emphasis on the comparison of the enzyme expressed in different tissues and developmental stages. Pu mutations have been induced by hybrid dysgenesis. Recombinant DNA cloning of the Pu region is proposed using P element and copia clones as probes to identify homologous inserts in the Pu region of the mutants, and to obtain Pu region DNA in cloned form. Restriction mapping of the region in wild type and mutant loci is proposed to define the functional region. Transformation of mutant embryos by the injection of DNA from the Pu region is proposed as a means of identifying the Pu locus. Methods for identifying the coding region by its homology to polyA+ RNA are also described. Biochemical and genetic approaches to the analysis of function in the non-structural loci affecting CH activity are discussed.
