The objective of this project is to analyze gene regulation for the argininosuccinate synthetase (AS) locus. The intent is to determine the molecular basis for metabolite regulation by arginine and for enzyme overproduction in canavanine-resistant (Canr) cells. The mechanism of enzyme overproduction in Canr cells may be related to the mechanism of high organ specific expression in liver. Structural analysis of the AS gene and multiple pseudogenes is well advanced and will be completed. Attempts are in progress to obtain expression of AS using DNA mediated gene transfer with cDNA clones and minigene constructions. Canr variants of heterozygous citrullinemia cell lines will be used to distinguish whethe the Canr phenotype is cis- or trans-acting. The primary method for analysis of regulation will be the use of DNA mediated gene transfer attempting to demonstrate (1) metabolite regulation with transfected DNA and (2) altered regulation associated with the Canr phenotype either related to the recipient cells or to the transfected DNA. Functional regions of transfected DNA will be identified using deletion mutants and linker scanning mutants. Experiments designed to clone regulatory genes are also proposed. The proposed experiments are directly relevant to understanding regulation of gene expression in mammalian cells. This remains a high priority endeavor in biomedical research. Analysis of this human locus has direct relevance to the disease citrullinemia, and regulatory mutants may exist in addition to the well known structural mutants.
