Regulation of human argininosuccinate synthetase gene: Induction by positive-acting nuclear mechanism in canavanine-resistant cell variants

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Abstract
Nonhepatic human cell variants resistant to the arginine analog, canavanine, express argininosuccinate synthetase (AS) mRNA at levels 200-fold higher than parental cells without amplification of AS gene sequences. In this report we show that this regulation occurs in the nucleus prior to polyadenylation of AS precursor RNA and occurs through a positive-acting mechanism operating in canavanine-resistant cells. The half-life of cytoplasmic AS mRNA was estimated by blocking cellular transcription with actinomycin D. In both parental and canavanine-resistant variants of RPMI 2650 cells, the AS mRNA decayed with a half-life of 12–24 h, showing that cytoplasmic mRNA stabilization was not involved in this regulation. Quantification of AS RNA following cell fractionation showed that AS precursor RNA was present at greatly elevated amounts in the nuclei of canavanine-resistant cells. Similar results were obtained when nonpolyadenylated RNA was examined. Thus, the mechanism underlying high expression of AS mRNA in canavanine-resistant cells is an early nuclear event, and the processes of polyadenylation and transport of RNA to the cytoplasm are not involved. Intraspecific somatic cell hybrids were constructed to test whether the induction of AS mRNA was due to a gain of a function in canavanine-resistant cells or to a loss of a function in parental cells. Quantification of AS mRNA in hybrid cell lines showed that such cells contained high levels similar to those found in the canavanine-resistant parent. These findings show that the induction of AS mRNA is due to a positive-acting mechanism operating in the nucleus of canavanine-resistant cells.