We have studied the regulation of expression of the
asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous
asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS
mRNA, and the stimulation of AS
mRNA expression is effectively suppressed by the addition of
asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the
mRNA as well as in the 5' genomic region. When a plasmid containing the human AS
cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS
cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of
asparagine, and this regulation required translation. When we introduced by mutagenesis a number of
stop codons in the AS
cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by
asparagine deprivation. Inhibition of
protein and
RNA synthesis also prevented down-regulation of AS
mRNA levels by high concentrations of
asparagine. In a parallel series of experiments, we showed that an AS
DNA fragment including the promoter and first exon can also regulate
RNA expression in response to
asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by
asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of
leucine,
isoleucine, or
glutamine. Thus, regulation of AS gene expression is a response to
amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.