Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line

Abstract

Previous studies of the human cell line RPMI-2650 (wild type) and its canavanine-resistant variants have demonstrated differences in argininosuccinate synthetase activity as follows: canavanine-resistant much greater than wild type grown in citrulline greater than wild type grown in arginine (Su, T.-S., Beaudet, A. L., and O'Brien, W. E. (1981) Biochemistry 20, 2956-2960). A recombinant plasmid containing a 1.55-kilobase insert complementary to the mRNA for human argininosuccinate synthetase was isolated by the combined use of differential colony hybridization and immunoprecipitation of the products of plasmid-selected mRNA translation. Both blot and dot hybridization analysis of polyadenylated RNA indicated a major mRNA species of 1.67 kilobase in all cells, and the levels of mRNA correlated well with the levels of enzyme activity: canavanine-resistant, 180; wild type grown in citrulline, 7; and wild type grown in arginine, 1. One major mRNA species of 1.67 kilobase and one minor species of 2.68 kilobase were observed in wild type and canavanine-resistant cell lines. Reassociation kinetics of pAS1 with genomic DNA from human liver, canavanine-resistant cells, and wild type cells were not significantly different. Blot hybridization of genomic DNA revealed no detectable differences between wild type cells, canavanine-resistant cells, and human leukocytes. The data demonstrated that there were multiple copies, perhaps 10 or more, of argininosuccinate synthetase-like sequences in human DNA and that the canavanine-resistant phenotype was not due to gene amplification.