Abstract
In order to study mRNA and DNA alterations in citrullinemia and to explore the basis of metabolite regulation and enzyme overproduction in cultured cells, we have isolated cloned cDNA for ASS. The human cell line RPMI-2650 (wild type, wt) was used to isolate canavanine resistant (Canr) variants which overproduce the enzyme. Activity of ASS in nmol/min/mg protein was as follows: wt cells grown in arginine medium, 0.14; wt cells grown in citrul-line medium, 0.86; and Canr cells grown in either medium, 25.0. Immunological studies indicated similar relative differences in amounts of enzyme antigen. A marked increase in translatable mRNA for ASS was demonstrated in Canr cells. A sucrose gradient fraction of mRNA from Canr cells was used to synthesize cDNA which was cloned in pBR322. A near full length cDNA clone for ASS was identified by differential filter hybridization and the identity confirmed by plasmid selected mRNA translation. Dot hybridizations and blot hybridizations after agarose gel electrophoresis demonstrated increased amounts of hybridizable mRNA proportional to the increase in enzyme activity in Canr cells. The patterns of restriction digestion of genomic DNA from wt and Canr cells were indistinguishable. The patterns were complex and suggestive of more than 1 or 2 gene copies. The data suggest that a regulatory difference between Canr and wt type cells allows a major change in mRNA accumulation without gene amplification.
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Bock, HG., Su, TS., O'Brien, W. et al. 702 CLONING OF cDNA FOR HUMAN ARGININOSUCCINATE SYNTHETASE (ASS) mRNA AND STUDIES OF ENZYME OVERPRODUCTION. Pediatr Res 15 (Suppl 4), 559 (1981). https://doi.org/10.1203/00006450-198104001-00725
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DOI: https://doi.org/10.1203/00006450-198104001-00725