The overall aim of this project is the investigation of the molecular mechanisms regulating expression of the arginase genes in normal man and in patients with hyperargininemia (liver arginase deficiency). Through gene transfer of our cloned liver and kidney arginase loci and flanking DNA regions into a variety of organotypic, arginase-expressing and -extinguishing cell lines, we will explore the genetic and cellular factors that are responsible for their unique tissue-specificity and differential induction. By in vitro directed mutagenesis, various footprinting methods, and DNA sequencing we will attempt to identify and precisely map those DNA sequences and/or trans-acting cytosolic factors necessary for conferring proper tissue specificity and inducibility by steroid hormones or substrate to the transferred minigene constructs, using, when indicated, a fused reporter genes. Through the molecular manipulation of signal sequences we will explore the importance of subcellular compartmentalization in physiologic function of the human arginase isozymes by redirecting the transferred enzymes into or out of the mitochondria. Transfer and expression of cloned arginase sequences from Macaca fascicularis, a primate species exhibiting a natural regulatory polymorphism for expression of AI in red blood cells, will be related to our broader findings of tissue- specific arginase regulation in man. Through these studies we hope to gain understanding of the basic cell biology of the human arginases, as well as widen our array of options for eventual gene therapy. The proposed research meshes well with my long-time interest in the regulation of gene expression, growing out of my undergraduate exposure to bacterial genetics and my graduate work on steroid hormone receptors as model eukaryotic gene regulatory proteins. It is also directly relevant to my clinical focus in medical genetics and mental retardation. I view the Clinical Investigator Award as essential in creating a secure and productive transitional environment to academic independence in which I can continue my fruitful association with Dr. Cederbaum and with the highly active and collaborative resources and training programs in molecular biology, medical genetics, and metal retardation that exist at UCLA.R
