SLOCUM Pyrimidine nucleotides are precursors of DNA and RNA biosynthesis and are constituents of activated intermediates in many metabolic processes which are fundamental to growth and development in plants and all other living organisms. The basic biochemical steps in de novo pyrimidine synthesis have been conserved in prokaryotes and eukaryotes but regulation of the pyrimidine pathway is accomplished by several different strategies involving subcellular compartmentation of enzymes and metabolites, gene duplication and gene fusion, and different metabolic control mechanisms. In plants, little is known about the mechanisms which regulate pyrimidine biosynthesis, but most evidence suggests that the important sites of regulation are the first and/or second steps in the pathway, involving the enzymes carbamoylphosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase). This research will focus on the elucidation of various molecular mechanisms regulating the activity of ATCase and CPSase in plants. As a first step toward this goal, cDNAs encoding three different ATCases in pea (pyrB1, pyrB2 and pyrB3) have been cloned. The pyrB1 and pyrB2 genes have been shown to be differentially expressed in pea tissues. These genes encode ATCase proteins whose primary sequences differ by 25% and it is likely that they have very different kinetic properties. and may participate to different degrees in the regulation of pyrimidine pathway activities. This possibility is being investigated, using a variety of approaches which includes the following: 1) kinetic characterization of the pyrB1 and pyrB2 products expressed in an ATCase-deficient strain of E. coli, 2) use of nucleic acid and antibody probes to investigate expression of the genes encoding ATCase in different plant tissues and in response to various treatments that would be expected to impact pyrimidine biosynthesis. Recently, the first plant genes encoding CPSase in Arabidopsis were cloned by the investigators. Availabili ty of cDNAs encoding the small and large subunit proteins of this plant CPSase (carA and carB genes, respectively) will permit the characterization of this gene and its expression in plant tissues. Expression of these CPSase proteins in appropriate E. coli CPSase mutants will facilitate biochemical and kinetic characterizations of the plant enzyme. Overexpression of the Arabidopsis CPSase proteins in bacteria will also facilitate their purification for biochemical studies and the production of CPSase antisera. These antisera, and the cDNAs encoding the plant CPSase proteins, will be invaluable tools in investigations of the regulation of CPSase and pyrimidine pathway activities. The investigators have now established that the basic genetic organization of the pyrimidine pathway in plants more closely resembles that of bacteria than other eukaryotes. Nonetheless, plants remain the only major group of organisms in which the fundamental control mechanisms regulating de novo pyrimidine synthesis have not been elucidated. Issues such as subcellular compartmentation of ATCase and CPSase activities, the kinetic and regulatory properties of the ATCases and CPSases themselves, metabolic interactions and regulation of pyrimidine and arginine pathway activities and many other questions will be addressed in this study. Studies such as these contribute to a broader understanding of plant primary and secondary metabolism and suggest strategies for the manipulation of plant biochemistry for many practical purposes, including the development of herbicides or other compounds that modify plant growth or development, and production of plant-derived pharmaceuticals and other useful metabolites. Apart from any purely scientific advances resulting from these studies, several undergraduate students will participate in the research. These students will gain valuable training in a modern research laboratory which will help to prepare them for post-baccalaureate careers in science.
