Research Project
6343018
Purines are fundamental biological molecules. They are components of DNA and RNA, the genetic information of all living organisms. Purines are critical as intracellular signaling molecules in the form of cAMP and cGMP, and are important intercellular signaling molecules functioning as neurotransmitters and as signaling molecules important for vasodilation and other physiological processes. ATP serves as a key molecule in energy metabolism. Many coenzymes are purine based. Genetic and biochemical regulation of purine synthesis in mammals and humans must be complex, involving several enzymatic steps and proteins. The pathway is almost certainly developmentally regulated in mammals and humans as well. These conclusions are based almost entirely on studies of the biochemistry of the pathway in whole cells or in cell free extracts. That such regulation must exist seems obvious. However, the molecular mechanisms of regulation remain to be defined. In no case has a detailed molecular analysis of the regulation of purine synthesis in an animal or in animal cells in culture been carried out. This is largely because until now the molecular reagents necessary have not existed. Without this information, it will be difficult to understand the role of the pathway in human disease and to intervene appropriately. The first seven steps of the pathway and genes encoding these are critically involved in the environmental, developmental, and genetic regulation of the pathway, and propose to use a novel set of reagents and capabilities, including human and Chinese hamster cDNA and genomic clones, antibodies recognizing functional domains of the proteins carrying out the fist seven steps and which are suitable for Western blotting, immuneprecipitation, and immunocytochemistry, and Chinese hamster ovary (CHO) cell mutants to understand these regulatory mechanisms. The roles of multi-enzyme complexes, multi functional proteins, transcriptional, translational, and posttranslational regulation of these five enzymatic steps in the regulation of purine synthesis will be evaluated. The following specific aims are proposed: (1) Determination of the nature and effects of mutations in CHO cells in the first seven steps of the pathway on the regulation of purine synthesis and purine levels; (2) Determination of transcriptional translational, and posttranslational regulation of these enzymes in response to alterations in environmental conditions using CHO cell cultures; (3) Determination of transcriptional, translational or posttranslational regulation of the first seven steps of the pathway during human and mouse development, (4) Assessment of the existence of multi-enzyme complexes involving the first seven steps of the de novo pathway and assessment of whether these complexes have regulatory significance.
