Regulation of argininosuccinate synthetase (AS) was studied by using minigenes containing 3 kilobases of DNA upstream from the TATAA box and 9 kilobases downstream (including the first four exons of the AS gene) ligated to either the cDNA for AS or to the chloramphenicol acetyltransferase (CAT) gene. Unlike the endogenous AS gene, expression of the CAT minigene was not elevated in Canrl cells, which overproduce AS compared with parental RPMI-2650 cells. Expression of the CAT minigene in both stable and transient analyses was four-to five-fold higher in RPMI-2650 cells grown in citrulline medium than in cells grown in arginine medium. Although endogenous AS activity is not subject to metabolite regulation in Canrl cells and expression of the CAT minigene in Can 1 cells was not increased when cells were grown in citrulline medium, expression of the CAT minigene was 10-to 22-fold greater when intracellular arginine pools were depleted by transient starvation for arginine and citrulline.Relatively few systems are available for the study of metabolite regulation of gene expression in cultured animal cells. Schimke (16) reported low levels of argininosuccinate cynthetase (AS) in cultured cells grown in medium with a nigh concentration of arginine. In contrast, AS activity was increased when cells were grown in medium with citrulline s:ibstituted for arginine or in medium with a low concentratiorn of arginine. Similar metabolite regulation of AS was observed in cultured human lymphoblasts (10, 11) and in a human epitheloid cell line, RPMI-2650 (18). A second aspect of regulation of AS in cultured cells involves overproduction of the enzyme in cell variants selected for resistance to the toxic arginine analog canavanine (11,18 (Fig. 1). The constructions used portions of the pSVOcat plasmid (8), which lacks the simian virus 40 viral promotor and enhancer elements. The CAT construction should synthesize authentic CAT protein rather than a fusion protein due to the presence of termination * Corresponding author. codons in the AS reading frame, to the 5' side of the CAT coding sequences. A small segment of DNA between the two HindIll sites in intron 1 was lost from both constructions.Expression of pMJ311 in Chinese hamster cells. The ability of the AS minigene to direct the synthesis of functional AS was tested in Chinese hamster cells, which do not express endogenous AS activity. RJK88 cells (7) were cotransfected with pMJ311 and pSV2gpt (14) by a modification of the calcium phosphate coprecipitation method (9). Stable transformants were selected either in citrulline medium or in HAT (hypoxanthine-aminopterin-thymidine) medium (7) to evaluate expression of AS with and without selection pressure. The activities were similar irrespective of the selection scheme (1.59 + 0.65 mU/mg of protein) and fell within the range of AS activity seen in cultured human fibroblasts (1). mRNA initiation sites. RNase protection experiments were performed to determine whether the CAT minigene transcripts were correctly initiated compared with...